Envisioning Homosexuality within Daoism -The Orientation and Sexual Dimensions of Yin and Yang
2015

D Bruce
Leblanc
Envisioning Homosexuality within Daoism -The Orientation and Sexual Dimensions of Yin and Yang

Int J Behav Res Psychol
38201510.19070/2332-Received: July 25, 2015 Accepted: October 13, 2015150 https://scidoc.org/IJBRP.php International Journal of Behavioral Research & Psychology (IJBRP) Citation: Bruce D. LeBlanc (2015) Envisioning Homosexuality within
Explicit examinations of homosexuality within Daoism have not been found within academic journals. This paper attempts to begin to address this issue. Drawing upon an understanding of the foundational principles of Daoism, including a yin/ yang and alchemical analysis, homosexuality is explored and analyzed within this spiritual/religious tradition. After reviewing these principles within a number of Daoist historical and schools of thought, homosexuality is examined from within Chinese historical and culture perspectives. Utilizing these combined perspectives this paper advances the position that homosexuality is inclusive of the principles of yin/yang and alchemical dynamics. A particular emphasis and analyze will examine the constructs of gender, sexual behavior, and sub cultural context and dynamics regarding how homosexuality incorporates the foundational principles and specific constructs of Daoism. Consequently, the position that homosexuality should be seen within the Dao is advanced.

Introduction

As we examine homosexuality within Daoism, with an emphasis on expanding the dimensional understandings of yin and yang, we must start by exploring the nature of humanity within this philosophical and religious tradition. This exploration will include brief glimpses into classic, alchemical, divinational, internal-alchemical and action and karma Daoism. After briefly exploring these foundations, including the dynamics of yin and yang, the nature of sexuality within the Daoist tradition is explored. These two foundations will be used for the comparative analysis of homosexuality and, through the use of Chinese historical references and contemporary Western frameworks, a reconciling integration of homosexuality. From these foundations an expansive application of yin and yang will be advanced.

Our foundational exploration of Daoism begins with The Classical Period (700-220 BCE) and an understanding of Dao (Tao), as "...an impersonal and unnamed forced behind the workings of the universe" [12]. The sages of this period embraced wu-wei, a process of"...going with the principles of the Tao" [13], which involved the lack of using force. Thus, sages were attempting to cultivate life and did so through physical and lifestyle/attitudinal practices. Physical practices involved breath work, calisthenics, and techniques involving the cultivation of sexual energy. Regarding lifestyle/attitudinal practices; desires, attachment, and activities that stimulate the mind, emotions, and senses advance ill health. In comparison to the benevolent Dao, of the Classic Period, the Dao of the Warring States period was more neutral, yet remained the origin of everything wherein "all" shared a common ancestry, and "....all things had equal standing in the universe" [14]. This led to the advancement of Chuang-tzu's [Zhuang zi] "principle of equality of all things" [11,14].

Since all originates and returns to the Dao (Tao), change, via motion, allows seeing patterns of change. Change is conceptualized as tai-chi, and in an older Wu-chi' [Wu qi] diagram there is a concentric halving of the yang (creation) and yin, (dissolution). Within this diagram of tai-chi, the inner circle is ancient yang and ancient yin, followed by greater yang and greater yin, with the circle farthest out from the center being lesser yang and lesser yin. For the Daoist yang and yin, as well as the pa-k'-ua [ba gua] and five elements are the foundational building blocks of everything. Regarding yang and yin, the I-ching [Yi jing] referred to these as complementary opposite pairs, including male and female. As opposites yin was seen as "....stillness, tranquility, softness, flexibility, female, and receptivity; and yang became associated with movement, activity, hardness, strength, male and initiative" [15]. Thus through movement and stillness yang and yin generate greater or lesser yang and yin within T'ai-chi [Tai ji]. Additionally, movement and stillness of yin and yang generates the pa-k'ua of ch'ien [qi ' This brief summarization of yin and yang and pa-k'ua [ba gua] helps with understanding alchemical Daoism (Taoism). While alchemical Daoism involves two forms of physiological alchemy, external and internal, it is internal alchemy that is more relevant to this presentation as it maintains that "....all the ingredients of immortality are found inside the body" [16], wherein the goal of internal alchemy is the transformation of the mind and body for longevity and health. Within this system it is extremely important to create and renew yin and yang via copulation, as well as through the application of how to use fire (yang) and water (yin) to cultivate energy while renewing life. Essentially, "life is renewed when impurities in the body are purged" [17]. One of the ways of cultivating energy was through sexual expression as noted by Tsan-tung-chi: "When ch'ien [qi'an] [sky or male] moves, it becomes erect. The vapor spreads as the generative energy flows. When k'un [kun] [earth, or female], is still, it contracts, becoming the furnace in the lodge of the Tao. Apply firmness, then withdraw. Transform it into softness to provide stimulation" [18]. Also within the alchemical tradition Wong notes Ko Hung's (P'aop'u-tzu) [Ge hong's {Bao pu zi}] position that physical techniques for the promotion of a long life must be accompanied by a correct mental attitude. Additionally Wong comments that during the Sung dynasty (960-1279 CE), Chen Hsi-yi advanced ideas, drawn from the I-ching [Yi jing] and Confucious, of cultivating virtue and circulating energy through physical techniques. Wong also recognizes Chang Po-tuan, also within the Sung dynasty, who "advocated the dual cultivation of the body and mind and combined methods of emptying the mind with physical techniques for circulating internal energy" [19], including the use of sexual techniques if undertaken in the early stages of body cultivation.

This alchemical Daoism is further expanded within the "way of transformation," advanced by internal-alchemical Daoism. Some basic ideas of this practice of Daoism include: "Before we were born, we were a part of the Tao. Formless and undifferentiated from the Tao"..... " When the generative energies of the father and mother come together, energy from the Tao is drawn into the womb of the mother to form a fetus".....and when the fetus is born.... "its internal energy is separated into three components: generative (ching), vital (ch'i [ji]) and spirit energy (shen)".......and thus " the practice of internal alchemy begins with realizing that the leakage of energy is the cause of many physical and mental problems in life" [20].

What is necessary is the recovery of the pre-birth energies, for one's health and longevity, and that occurs through internal transformations. Through the utilization of various fields of energy in the body (The Furnace, the Cauldron, and the Tan-t'iens [Dan tians] (elixir fields)), energy is transformed. In the lower tan-t'ien [dan tian], the region of the naval, generative energy is transformed into breath energy, which is then transformed in the middle t'ien [tian], located in the heart region, into spirit energy. This spirit energy is then transformed in the upper t'ien [tian], located between the eyebrows, into "the primordial vapor of the Tao" [21].

The transformative process within internal-alchemical Daoism involves adjustments to the fires in the tan-t'iens [tians], thus there is a need to know when to utilize fast or slow fires as one utilizes yang and yin fire. Regarding the nature of these fires "yang fire refers to fast breathing and it is used to direct the fire to the middle and upper tan-t'iens [tians]. Yin fire refers to soft, slow breathing and it is used to incubate the internal energy" [22]. The interplay of yang and yin energies is also seen in the principle of the Copulation of the Dragon and Tiger, where yin is the dragon and yang the tiger. The copulation or union of yin and yang is seen through body symmetry where the "....back of body is yang and the front in yin; the left side of the body is yang and the right side yin; the upper body is yang and the lower part yin" [22]. Unification of these energies is accomplished through the dissolution of barriers and blockages, which allow the energies of yin and yang to meet and be transformed in the internal cauldrons.

Within internal-alchemical Daoism this process involves the activation of three stages, the first which builds foundations, the second that transforms the internal energies, and the third that "completes" the alchemical works. The first stage involves external (wai-chuang) strengthening, focusing on the muscles, ligaments, joints, and spinal column; as well as internal strengthening (neichuang), that massages the internal organs and stimulation of the nervous system. The final process in the first stage is refining the mind (lien-hsin) to still the mind, minimize desire, and develop an uninterested mind regarding excitement and sensual stimulation. The middle stage (lien-ching-hua-ch'i) "focuses on gathering, refining, and transforming generative energy" [23]. One method for transforming generative energy is through paired sexual activity. In the paired sexual activity generative energy can be gathered from one's partner. Yet, for the generative energy to be available for utilization the sexual acts must not be procreative or involve sexual pleasure or desire, as either of these depletes the generative energy. The generative energy is then refined in the furnaces, by regulating breath, sealing cauldrons so energy will not be drained, and transforming energy (ch'i [ji]) into vital energy. The refinement of ch'i [ji] needs to minimize changes in one's emotional state, including the avoidance of anger, fear, sadness and frustration. The internal alchemical work (lien-shen-huan-hsu) wherein ch'i [ji] is refined in the upper tan-t'ien [dan tian], involves the emptying of thoughts within the mind, extinguishing the duality of object and subject, and "being in a state of total emptiness" [11,24]. Thus there is the transformation of the three energiesgenerative, vital and spirit, to "their original undifferentiated state is the emergence of the seed of the Tao" [24].

The Action and Karma School of Daoism provides another relevant foundation for the topic at hand. This school is concerned with everyday issues and right ethical actions. The foundations of this school maintain that good acts are harmonious with the Celestial Way, while malevolent acts are opposed to the Celestial Way. Thus, as noted by Wong, T'ai-p'ing ching maintains that one should "accumulate good deeds, and property will come from the Tao" [25]. Essentially, good actions can mitigate that which causes illness, while doing wrong can encourage the development of illness. Two relevant beliefs of this school of Daoism are: "For every action, there is a response from the Tao" and "....rewards can be health and longevity as well as wealth and prosperity, and retribution can be illness or shortening of the life span as well as poverty and misfortune" [11,28].

Having provided some foundations regarding sexuality within the Daoist perspective, it is important to provide a brief review, and thus understanding regarding homosexuality within Chinese culture, as well as within Daoism. For this investigation we will draw upon two major works, the first written by Dr. Fang Fu Ruan Dr. Ruan presents what is understood about classical Chinese sexology even though most of the formal classical texts have been lost. Ruan comments that there is a relationship between classical sexology, traditional Chinese medicine, and Daoist sexual techniques, all of which have a common foundation based on "the principles of Yin and Yang, the five elements, and the concept of chi (vital energy)" [28]. One important concept within Chinese sexology is the "seven injuries and eight advantages" (qui sun ba yi /qi qun ba yi) [27]. It is through this concept that the two principles (Yin and Yang) "...can be brought into harmony" [27]. Furthermore, Ruan highlights the Methods of Intercourse between Yin and Yang, which describes in great detail the process of not only intercourse, but foreplay, a process that involves vital energy (chi), in such a manner that the latter may be sufficiently arousing that "consummation" is not necessary. Yet, through sexual intercourse there is a movement of chi that is balanced between that which is hard (penis) and soft (vagina), which promotes the "natural way of Heaven and Earth" [28]. The naturalness of sexual activity is essential as and supported when Ruan cites Ge Hong, "It is inadmissible that man should sit and bring illness and anxieties upon himself by not engaging in sexual intercourse. But then again, if he wishes to indulge his lusts and cannot moderate his dispersals, he hacks away at this very life" [28]. Although one must not be lustful, this sexual naturalness need not be confined to one partner, as "changing of partners can lead to longevity and immortality. If a man unites with one woman only, the Yin chi [vital energy] is feeble and the benefit small" [28]. Even though one can have sex with more than one person, within these encounters it is the exchange of vital energy that is essential, because if the male has no emission he will remain fit and his complexion perfect into old age. This latter behavior is the central component, or essence, of the Art of the Bedchamber. So important is sexual activity that Ruan cites Pheng Tsu [Peng zu] in On Delaying Destiny who commented, "....man does not want to be without women; if he has to do without her his mind will become restive, if his mind becomes restive his shen (spirit) will become fatigued, and his life-span will be shortened" [29]. Yet, Ruan notes Tsu's comment that if a man could remain serene, not being bothered by sexual thoughts, that would be ideal, yet he believes that "....there is not one among ten thousand who can do it" [29].

Regarding homosexuality within Chinese society and culture, Ruan recounts that stories regarding homosexuality can be found in "The Difficulties of Persuasion" which includes the story of king Ling and his love for Mi Tzu-hsia [Mi ziheng]. Mi violated a law by taking the carriage of the king but was not punished for his act but rather praised for his "filial piety." Additionally, Mi saved half a peach for the king, for which the king acknowledged Mi 's love publically, which led to the expression "sharing the remaining peach" (yu-tao). A second story involves the Emperor Han Ai-ti (6 B.C.-1 A.D.) who loved Dong Xian and while the two were napping together Emperor Ai-ti cut his long sleeved gown so as not to disturb Xian's sleep. This gave rise to the usage of "the cut sleeve" (tuan hsiu). Both yu-tao and tuan hsiu refer to homosexuality. Beyond these imperial stories, homosexuality was known and accepted during the Zhou and Han Dynasties, while also being seen in the Ming and Ching Dynasties. During the Zhou Dynasty one story involved Qi Jing Long, who received the glance of an officer because of his beauty. The king noted that if the officer had sex with the king, the death penalty would result. Yet, a premier, Master Yen, informed the king that"....it was not right to reject sexual desire, and not good to reject love" [30]. Also during this dynasty homosexual seduction was used as a weapon of the military or politicians, as it was believed "...a beautiful man can seduce an old man" [30]. Ruan goes on to note that during the Han Dynasty ".....ten of the eleven emperors had at least one homosexual lover or expressed some homosexual proclivities" [31]. More significantly Guo-ti, the founder of the Han Dynasty, had a homosexual relationship with Ji-ru; while Wu-ti, the Marital Emperor, had male partners including Han Yen, Wei Qing, and Huo Chu-bin, the former a court musician and while the latter two were generals. Outside of these social elites it was during the Ching Dynasty that homosexuality was seen beyond this social group. During the Ching Dynasty the status of shialmg gung/ gu emerged. This status referred to males acting as females, or to the male homosexual's lover. An occupation was associated with the status of shialmg-gung, which flourished, was where males dressed as females and had male admirers. Another homosexual outlet during the Ching dynasty was providing younger male monks, for a significant fee, to powerful men [8].

From this foundational discussion of Daoism and then the specific related understanding of how sexuality and homosexuality have existed within Chinese culture and Doaism, a sufficient background has been provided to undertake an applicational analysis that will propose an expansion of the application of these concepts in a affirmational manner to homosexuality within Daoism. More specifically, the expansion of the understandings and applications of yin and yang, in an affirmative manner, will be argued.

When one looks holistically at the brief presentation of the Dao, Yin and Yang, sexuality and homosexuality, the expanding of Yin and Yang within the Dao and Daoism must be carefully constructed. Recognition must be given to the historical understandings of Yin and Yang within the framework on not only chi but also chi within not only individual men and women, but relationally between men and women. As such it must be acknowledged that the normative state of Yin and Yang and thus male and female is a complementary relationship, as highlighted by Drs. Ruan and Chang. Thus within the Daoist framework the nature of not only the "natural" relationship for humans, but also the "natural" sexual relationship for humans would be that of male and female who embrace and share the chi of life through their utilization of their dominant yang and yin, respectively. Yet, as noted by Chang, each sex is imbued with both Yin and Yang. Thus the traditionally Daoist visual portrayal of yin and yang encapsulates this reality. This position is supported not by research regarding Chinese and Daoist relationships and sexuality, but rather is supported by research within the Western traditions, most notably the National Health and Social Life Survey (NHSLS) [2]. This research found that about ten percent of males and eight percent of females identified themselves behaviorally, by desires, or through direct self-identification as homosexual. Thus nearly ninety percent of the population identifies themselves as heterosexual. Consequently, the "normative" nature of complementary pairing would be that of male and female and might be visually conceptualized as occupying most of the symbol for Daoism, while conceptualizing homosexuality within this symbol would occupy only a portion of the Daoist symbol. While acknowledging heterosexuality as normative, this dominant complimentary pairing of the opposite sexes being attracted to one another within heterosexuality is not scientifically substantiated. Essentially, the causes of the sexual orientation of heterosexuality have not been adequately researched in humans. https://scidoc.org/IJBRP.php Now as we transition and expand the discussion to include homosexuality one first must confront the question raised by Yuan Mei and Chi Yun of the nature versus nurture positions. Yuan Mei, a famous Ching Dynasty author, relates a story of a man who appeared more feminine and did not get along with his wife. He was a man who was portrayed as wanting to be more of a man's concubine than a husband to his wife. This man was compared to a tree, that has many different branches, and therefore that this state of his being was something he was born with, just was a tree has its branches. In contrast Chi Yun in his writings noted that lusting after women was part of the "natural sexual drive," while desiring a catamite was not. He believed such lusting or desires for catamites came about from socialization or seduction processes. Thus Mei represents the "nature' position, while Yun represents the "nurture" position regarding the origins or causes of an individual's homosexuality.

To respond to the positions posed by Mei and Yun, we again must draw upon Western research since within the Chinese and/ or Daoist positions there has been no significant research endeavors to answer the nature/nurture question. While the question of nature or nurture has not been "answered" within the Western tradition, a few research studies provide some support for a nature position. The first of the studies involves the examination of identical twins. This study found that if one male was homosexual in identical twins that over one half, fifty-two percent, of the other twins were homosexual [10]. A second study, conducted by the same researchers, found that the same finding was true for female homosexuals. Thus the correlational conclusion was that homosexuality is a heritable characteristic. In a more physiological study Simon Levy found that an anterior portion of the hypothalamus, which is agreed upon or understood to be related to sexual drive (towards the object of attraction/desire), is anatomically similar in size for heterosexual women as it is in homosexual men, while the same area of the hypothalamus is larger in heterosexual men, as it related to sexual orientation [5]. While these findings do not prove that homosexuality, either as a sexual orientation or behavior, is biological and thus of "nature," such findings cannot be dismissed. Both the genetic connection supported by the twin studies, and the organic connection found in the Levy study support a "nature" position regarding homosexuality. Consequently, the nature position must be considered not only as part of Western culture but also Eastern and Asian culture and Daoism.

Thus it can be argued that homosexuality within the Eastern and Daoist framework, as supported by this nature position and because of the noted genetic and organic findings, is part of the Dao. It would not only be part of the universe, but would also share the common ancestry of Dao, as conceptualized during the Warring States period. Furthermore, if something has a natural genesis, not merely behavioral, as conceptualized by the alchemical Daoist, homosexuals and homosexual behavior would have to be seen within the context that "before we were born, we were all part of the Tao" [20] and thus a natural human condition, albeit for a smaller portion of humanity. The alchemical Doaists, through maintaining that "all the ingredients of immortality are found inside the body" can also be used as a supportive context for the innate naturalness of homosexuality because of these organic and genetic findings.

The support for a nature position also can draw upon the writings of Ko Hung and the construct that long life needs to be connected with "a correct mental attitude." Thus a homosexual male, at least as seen in Levy's study, who has a portion of the brain that is associated with sexual drive, and secondarily sexual orientation, that is correspondingly like that of a female heterosexual, would need to act in accordance with their physiology. In addition their correct mental attitude would be the enactment of the internal drives of that region of the brain. To act contrary to those drives would not be enacting the appropriate mental attitude. Additionally, drawing upon the action and karma school of Daoism, one must "accumulate good deeds" and such good acts are "harmonious with the Celestial Way." Conceptually, how could one's denial of the physiological nature of their sexual orientation, in this case homosexuality, and any actions resultant of that denial be consider "good deeds?" Such evidence of this denial is seen within the homosexual community by individuals who embrace heterosexual relationships for periods of their life and then either have extra, relational/marriage affairs or by "coming out" during their mid-life. The damage that is inflicted upon their partners cannot be considered a "good deed," nor is it "harmonious," not to mention the internal disharmony such actions cause. The eventual "coming out," at least from a Daoist perspective, could be conceived as a "response from the Tao," that will not allow the internal nature of the individual to live a disharmonious life, nor continue to undertake "bad" deeds. Thus if one is to live a life that promotes harmony, internally and externally, one must live a life that allows one's ch'i [ji] to be enhanced, through the refinements previously noted, and as such would need to minimize negative emotional states, such as frustration or sadness. Not being true to one's physiology, through internal or external denial, can promote such negative emotional states. Consequently, ch'i [ji] would not be enhanced and one's life and health would be impacted. Additionally, Pheng Tsu's [Peng zu] comment that if man does without a woman that his mind is restive, and if restive the man will have a fatigued spirit and shortened life, although specifically noting a heterosexual pairing, can be applied to homosexuals as well. If the homosexual does not enact that which their physiology is "calling forth," then their minds can also become restive, just as is proposed for heterosexuals. If restive they correspondingly have the potential to create a fatigued spirit and a shortened life. Therefore to maintain one's spirit (shen) the homosexual should embrace the seeking out of their desired partner, as any demand for living a non-sexual life, although an ideal (for heterosexuals), seems unlikely. Remember that Tsu noted that not one in ten thousand might be able to embrace such a decision.

Beyond this purely genetic and organic orientation, this position also embraces the essential elements of yin and yang. Remember that within Chinese medicine both yin and yang are necessary and that men and women have both, even while one is dominant within each sex. Not only are they necessary as Chang commented, because of the hormonal dimensions of yin and yang, men and women can, and sometimes desire, to express the masculine and feminine dimension of either in their lives. Not only are there expressions of yin and yang in males and females, there remains that complementary dimensions, and resultant balances that exist between the sexes. So how does the physiology of yin and yang and any complementary nature of these "energies," apply to homosexuality. Before commenting on the any applicability to yin and yang, a brief examination of any complementary dimensions of homosexual relationships is warranted.

Expressions of masculinity and femininity have been researched in relationships, including homosexual relationships. For heterosexual couples the consistent findings, albeit with some younger https://scidoc.org/IJBRP.php generational changes, hold that specific household takes and gendered behaviors are undertaken and enacted by males or females in these relationships. Through limited research the same seems to holds true for homosexual relationships. Five research studies [4,6,7,9] found that an enactment of predominantly either male or female instrumental or socio-emotional roles was undertaken by the respective partners in female and male homosexual relationships. In the homosexual relationships studied complementary roles were enacted in these relationships where one partner enacted male identified roles, while the other partner enacted female identified roles. Such role enactments can also extend into the realm of intimacy and sexual behavior. Within the female homosexual community this can be seen when a "butch" female is in a relationship with a "lipstick lesbian," the former embracing more masculine traits, while the latter is extremely feminine. While, within the male homosexual community the complementary gendered relationship would be that of a "macho,"or masculine male and a "femme," or feminized male. Sexually this is seen in homosexual relationships where one is more of a "top" and the other a "bottom." The top plays the more assertive, and/or insertive, role in the sexual encounter. For a female homosexual relationship this might involve the use of a "strap-on dildo," while in a male homosexual relationship it would involve genital insertion, the former into the vagina and the latter into the anus.

This complementary social behavioral dimension of homosexual relationships provides the context for supporting the enactment of yin and yang within these relationships. The enactment of these complementary gendered roles is viewed as an expression of yin and yang, within the lipstick/butch and femme/macho homosexual relationships respectively. The lipstick female and femme male respectively would display the yin social characteristics of being tranquil, soft and flexible; while the butch female and macho male would display the yang social characteristics of movement, activity, and strength.

Regarding the application of yin and yang to the sexual dimensions of a homosexual relationship the engagement of the internal furnaces, including through sexual activity, for the refinement of ch'i [ji] is utilized. Recall that Ge Hong noted that it would be wrong for a man to become ill and anxious by not engaging in sexual intercourse, but must do so without indulgence, although it is acceptable with multiple partners (at least for men). Also remember that Tsan-tung-chi vividly describes sexual intercourse as a means of renewing life by creating vital energy. Thus the first conclusion, whether one embraces the nature or nurture position, is that sexual activity is necessary for one's life, including one's ch'i [ji]. Consequently, in intercourse the exterior yang enters the interior yin, thus promoting intersexual balance. As it applies to homosexual couples, the "top" male with yang energy would enter the "bottom" male with this yin energy; while for female couples the natural sharing of energies is not facilitated by the use of the inanimate dildo, as noted by Chang, but can still be facilitated through the use of one's ch'i [ji] that is shared through digital and/or oral stimulation by the "dominant" female to the "receptive" female in the relationship. Both enactments were recognized by Chong and reiterated here.

Also within the context of sexual expression one can use breathing to enhance the energies of yin and yang, as advanced by internal-alchemical Daoism. This is most readily accomplished though the sexual response cycle wherein breathing for the active partner can escalate and deepen, particularly as they reach orgasm. As such the active and/or insertive, partner would be using breath work to facilitate the development of their yang energy, if done with the intentions to fuel the internal fires. Contrastingly, the passive and/or receptive partner could breath slowly thus incubating not only their internal energies, but also the energies shared through the sexual experience. Thus if one has intentionality regarding the utilization of sexual practices to enhance one's vital energy, through right intention and mind, it is proposed that one can have a magnification of vital energy within such homosexual sexual encounters.

Finally, for physical behaviors beyond the potential energy dynamics of penetrative sexual activity, foreplay (as noted by Ruan), if sufficiently arousing and because it involves ch'i [ji]or vital energy, can be enough that sexual endeavors need not progress to sexual intercourse. Thus within homosexual parings, even beyond any aforementioned penetrative behaviors, these individuals are certainly capable of engaging in foreplay and thus can stimulate vital energy for themselves and their partners.

Yet physiologically there is a further dimension of yin and yang that can be analyzed within a homosexual relational context that involves the positions of the body during sexual behaviors that might facilitate the flow and enhancement of vital energy or ch'i [ji] between partners. Here it is necessary to recall the internalalchemical Daoist position that yin and yang can be connected via body symmetry. Recall that this symmetry involves the front of the body (yin) and the back of the body (yang) and the upper body (yang) with the lower body (yin). Therefore within a sexual behavioral context homosexuals as well can engage in behaviors that enhance this symmetry. More specifically within homosexual male sexual encounters if the "top" male enters the "bottom" male from a rear entry position, their front would be encountering their partners back. As such the yang of the "bottom" would be encountering the yin of the "top" in a context that has the more gendered male "top" with their yang, also interacting with the female gendered male "bottom's" yin. Thus there are two complementary interactions of yin and yang. Another example would involve oral sexual behaviors where the upper body of one partner encounters the lower body of the other partner. This most certainly occurs when couples, of any sexual orientation, engage in oral sexual stimulation. The upper body yang intersects with the lower body yin in both fellatio and cunnilingus. Again the gendered dimensions of the argument can be applied to both male and female homosexual couples, particularly when it is the gendered "male" that is performing oral sex on their gendered "female" partners. Therefore within these sexual acts, if done with "mindful" intention for the enhancement of ch'i [ji], the complementary dimensions and interactions of yin and yang can be utilized and facilitated through these sexual acts.

Another dimension of a complementary dimension of homosexual relationships can be seen in the multigenerational or economic dynamics seen within some homosexual couples, which are viewed as expressions of yin and yang. The most visual complementary pairing is when an older person dates a significantly younger person. One conceptualization of this within the male homosexual community is the "daddy"/ "son," (of legal age) pairing. This type of relationship is multigenerational with the older partner generally being at least fifteen to twenty years older than the younger partner. Within these relationships the older partner brings a social, economic, and often psychological stability, and consequently, within the context of this paper, more yang to the https://scidoc.org/IJBRP.php relationship. In contrast the younger partner draws upon this social and psychological stability and can provide nurturing to the older partner. Thus the younger partner, within the context of this paper, expresses more yin in the relationship. An examination of over five hundred online profiles from mainland China, at a sight dedicated to multigenerational homosexual relationships, found a vast majority of these individual seeking to find such an older male partner. Additionally, over three hundred of these individuals indicated that one of their preferences was to be the "bottom" within the sexual relationship with these older partners. Thus not only is the complementary nature of yang and yin desired within the social gendered relationship, it is also specifically sought out sexually within the desired relationship [3]. Again, while not establishing causation, these multigenerational pairings of contrasts provide additional support for a complementary relationship that because of these dynamics can facilitate the yin and yang of both individuals. As such, these relationships provide further support of the naturalness of homosexuality as it relates to the Dao.

Conclusion

Although there has been some affirmation of homosexuality within the Chinese and Daoist traditions, this affirmation has been somewhat limited regarding the integration of contemporary scientific and cultural research. This analysis attempted to expand and deepen an affirmation and integrative position for homosexuality within Daoism, with a particular reflection, and expansion, of yin and yang to this sexual orientation; while recognizing that such a position is outside the traditional Daoist position of he yin/yang and male/female and the creation of life. This affirmational position was accomplished through the use of not only more current physiological research, but also cultural research.

While certainly not an exhaustive analysis of these concepts with an application to homosexuality, this analysis provided a clearly articulated position regarding how homosexuality can be affirmatively and integratively supported within Daoist. The physiological and genetic evidence provides the most supportive evidence for this position. While not scientifically causative, the strong correlative implications within a physiologically agreed upon location of biological and/or psychological drives in the hypothalamus, cannot be dismissed. This combined with the genetic heritability seen in identical twins provides further evidence to support a nature position regarding homosexuality. Consequently, if a nature position is supported, even with this correlational evidence, this sexuality orientation must be seen as of nature within the Dao.

From this physiological and genetic foundation a direct application, and expansion, of how yin and yang can be viewing within homosexual sexual and social relationships was argued. Just as with Western culture mere historical understandings and frameworks for understanding yin and yang must be examined. A detailed and potentially graphic application of yin and yang was presented regarding not only homosexual sexual behaviors, but also complimentary social pairings seen within segments of the homosexual community.

Combined the physiological, genetic, and complimentary pairings seen within homosexuality and the articulated applications of yin and yang provide a clear theoretical position for the naturalness of homosexuality within Daoism. From this theoretical position further discussion can and should be undertaken regarding the nuances of homosexuality within Daoism and in particular how yin and yang can be cultivated within such relationships.

titled Sex in China, Studies in Sexology in Chinese Culture; while the second was written by Dr. Stephen Chang entitled, The Tao of https://scidoc.org/IJBRP.php Sexology-The Book of Infinite Wisdom.

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Norartocarpetin from a folk medicine Artocarpus communis plays a melanogenesis inhibitor without cytotoxicity in B16F10 cell and skin irritation in mice

Horng-Huey Ko
Yi-Ting Tsai
†
Ming-Hong Yen
Chun-Ching Lin
Chan-Jung Liang
Tsung-Han Yang
Chiang-Wen Lee
Feng-Lin Yen
Norartocarpetin from a folk medicine Artocarpus communis plays a melanogenesis inhibitor without cytotoxicity in B16F10 cell and skin irritation in mice
R E S E A R C H A R T I C L E Open Access
Background: Many natural products used in preventive medicine have also been developed as cosmeceutical ingredients in skin care products, such as Scutellaria baicalensis and Gardenia jasminoides. Norartocarpetin is one of the antioxidant and antityrosinase activity compound in Artocarpus communis; however, the cytotoxicity, skin irritation and antimelanogenesis mechanisms of norartocarpetin have not been investigated yet. Methods: In the present study, cell viability in vitro and skin irritation in vivo are used to determine the safety of norartocarpetin. The melanogenesis inhibition of norartocarpetin was determined by cellular melanin content and tyrosinase in B16F10 melanoma cell. Moreover, we examined the related-melanogenesis protein by western blot analysis for elucidating the antimelanogenesis mechanism of norartocarpin.Results:The result of the present study demonstrated that norartocarpetin not only present non-cytotoxic in B16F10 and human fibroblast cells but also non-skin irritation in mice. Moreover, our result also first found that norartocarpetin downregulated phospho-cAMP response element-binding (phospho-CREB) and microphthalmia-associated transcription factor (MITF) expression, which in turn decreased both synthesis of tyrosinases (TRP-1 and TRP-2) and cellular melanin content. This process is dependent on norartocarpetin phosphorylation by mitogen-activated protein kinases such as phospho-JNK and phospho-p38, and it results in decreased melanogenesis.Conclusion:The present study suggests that norartocarpetin could be used as a whitening agent in medicine and/or cosmetic industry and need further clinical study.

Background

In recent years, natural products and medicines have been developed as cosmeceutical ingredients to resolve esthetic skin problems such as skin darkening and wrinkle [1][2][3][4]. Artocarpus species, including A. heterophyllus, A. lakoocha, A. communis, are Asian or Pacific tree crops that are commonly used in agriculture, traditional medicine, and industry [5]. Artocarpus species have been shown to possess many pharmacological properties, which include anti-inflammatory [6,7], tyrosinase inhibitory [8], antitumorigenic [9], antidiabetic [10], antibacterial [11], antitubercular [12], antiviral [13], antiplatelet [14], and antioxidant activity [15]. Some of these effects might be due to the antioxidant and anti-inflammatory activity of norartocarpetin (2-(2,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one; Figure 1), a flavonoid compound present in A. communis and A. heterophyllus [7]. However, the biological pathways that norartocarpetin targets have not yet been fully investigated.

Normal melanin production is required to prevent ultraviolet (UV)-induced DNA damage since it absorbs UV radiation and lowers the occurrence of skin cancer [16,17]. Unfortunately, skin color darkens as a result of excessive exposure to UV radiation due to activation of the alphamelanocyte stimulating hormone (α-MSH) pathway, which results in melanogenesis [18]. α-MSH, a cyclic adenine monophosphate (cAMP) elevating agent, is usually used to induce the phosphorylation of cAMP response elementbinding protein (CREB) and enhance microphthalmiaassociated transcription factor (MITF) protein levels [19]. Previous studies have demonstrated that MITF is the major regulator for synthesized tyrosinase (TYR) and its related proteins (TRP-1 and TRP-2) [20,21]. These tyrosinaserelated proteins are the rate-limiting enzyme of melanogenesis since they regulate conversion of tyrosine to dopaquinone, rearrangement of DOPAchrome to 5,6-dihydroxy-indole-2-carboxylic acid, and abnormal accumulation of melanin pigments. In addition, phosphorylation of mitogen-activated protein kinases (MAPK) and signaling cascades of extracellular responsive kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 also modulate melanogenesis [22,23].

Therefore, skin whitening agents can inhibit MITF transcriptional activity by decreasing TYR protein levels through MAPK-mediated MITF phosphorylation. The MAPK-mediated MITF degradation pathways activated by norartocarpetin have not been investigated yet. The aim of this study was first to determine the toxicity of norartocarpetin in vitro and in vivo model and then to define the pathway by which norartocarpetin inhibits the melanogenesis signaling cascade by examining the activation of MITF transcription regulators (p-CREB, MITF, TYR, TRP-1, and TRP-2) and phosphorylation of MAPK signaling pathways (p-ERK, p-JNK, and p-p38).

Methods

Chemicals and reagents

Norartocarpetin purification

The heartwood of A. communis was obtained from Tainan district agricultural research and extension station, Council of Agriculture, Taiwan.  dried extract (160 grams) was then dissolved with equal volume of dichloromethane (DCM) and ethyl acetate (EA). The EA partition was subjected to silica gel column chromatography and eluted with different proportions of n-hexane/EA (19:1 and 8:2) collected solution was then eluted with an equal proportion of DCM/EA and DCM/ acetone (2:1). The fraction was then purified on a Sephadex LH-20 column to obtain norartocarpetin. Norartocarpetin is a light yellow powder. The UV spectrum of norartocarpetin in methanol showed absorption maxima at 263 and 350 nm. The IR spectrum showed hydroxyl, conjugated carbonyl and aromatic ring absorption bands at 3071, 1661 and 1619 cm −1 , respectively. The electrospray ionization mass spectrometry (ESIMS) of norartocarpetin gave a [M + H] + peak at m/z 287 and a [M + Na] + peak at m/z 309, which corresponded to a molecular formula of C 15 H 10 O 6 . The structure of norartocarpetin ( Figure 1) was also determined using NMR. The NMR data is as follows: 1

Cytotoxicity of norartocarpetin

B16F10 melanoma cells and human fibroblast cells (Hs68 cell line) were purchased from BCRC (Bioresource Collection and Research Center, Hsinchu, Taiwan), which originally purchased them from ATCC (USA). B16F10 melanoma cells were cultured in complete DMEM (Life Technologies, USA) (10% fetal bovine serum, 100 units/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B) in an incubator at 37°C with 5% CO 2 . Briefly, 1 × 10 4 B16F10 cells and human fibroblast cells were seeded in 96well culture plates and allowed to adhere for 24 h. After adhesion, a series of norartocarpetin concentrations were dissolved in DMSO, diluted in DMEM medium, and added into each well for 48 h. At the end of the incubation, the residual medium was removed, and 150 μl of 5 mg/ml MTT solution was added to each well and incubated for 4 h at 37°C. The medium was removed, and 100 μl DMSO was added to each well, which was then gently shaken. The 96well plates were then quickly measured at 550 nm with a microplate spectrophotometer (BIOTEK, μQuant, USA). The absorbance of cells treated with DMSO was considered the control and compared with that at different norartocarpetin concentrations. All determinations were performed in triplicate.

Skin irritation of norartocarpetin

The evaluation of skin irritation is the major index of dermal safety in cosmetic application and therefore the dermal safety of norartocarpetin was conducted according to the Draize test described by ISO-10993-10 (Tests for irritation and skin sensitization. The protocol has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Medical University (Approval number: IACUC-98146). Briefly, four male hairless mice, 6 weeks old BALB/c Nude mice (20-25 g), were obtained from the National Laboratory Animal Center, Taiwan. Mice were acclimatized and fed with a standard rat chow diet and water ad libitum in specific pathogen free laboratory for one week. All mice were received humane care in accordance to the "Guide for the Care and Use of Laboratory Animals" (National Academies Press, Washington, DC, USA, 1996). Norartocarpetin was dissolved in vehicle solution (propylene glycol 400:ethanol = 7:3 v ⁄ v) for external administration. The dorsal skin of mice was divided to four test sites (approximately 1.5 cm × 1.5 cm) for application and observation. The test period was three days and each mouse was topically treated once daily with 50 μl of vehicle solution, 0.02%, 0.1% and 0.2% norartocarpetin formulation in four test sites, respectively. The appearance of each application site was recorded at 24 h, 48 h and 72 h following external administration. The skin irritation of test sample, such as erythema or edema, was evaluated by the scoring system of Draize test, including (0): no erythema or no edema, (1): very slight erythema or edema, (2) well-defined erythema or edema, (3): moderate erythema or edema, (4): severe erythema or edema.

Determination of cellular melanin content

Cellular melanin content was determined as described previously, with only slight modifications [24]. Briefly, 1 × 10 5 B16F10 cells were seeded in 6-well plates and cultured at 37 overnight. B16F10 cells were then treated for 48 h with various concentrations of norartocarpetin (0.01-10 μM). Cells were then washed with PBS twice and lysed in 150 μl of 1 M NaOH. The lysate was heated at 95°C to solubilize the melanin, and then, 100 μl lysate was added in 1 well of a 96-well microplate. The plate was then quickly measured at 490 nm with a microplate spectrophotometer (BIOTEK, μQuant). In addition, we also tested the antimelanogenesis activity of norartocarpetin on α-MSH-induced melanogenesis. For this, B16F10 cells were treated with various concentrations of norartocarpetin for 24 h and then with 10 nM of α-MSH and incubated for 48 h. The determination of melanin content was performed as described above. All determinations were performed in triplicate.

Determination of cellular tyrosinase activity

Cellular tyrosinase activity was measured as previously described [25], using a culture method similar to the melanin content assay. Briefly, the wells were treated with norartocarpetin in the presence or absence of 10 nM α-MSH for 48 h. Cells were then detached with trypsin-EDTA and centrifuged for 10 min at 12000 rpm in order to obtain cell pellets. The pellets lysed with 100 μl 1% Triton X-100 and 100 μl 0.1 mM PBS (pH 6.8) containing phenylmethylsulfonyl fluoride. The cell lysate was then frozen and thawed twice before being centrifuged for 10 min at 12000 rpm. The supernatant (80 μl) was added in a 96well plate and mixed with 20 μl 0.2% L-DOPA. After incubation for 1 h, optical densities were measured at 475 nm using a microplate spectrophotometer (BIOTEK, μQuant). The inhibitory activity of the norartocarpetin-treated cells is presented as a percentage of the untreated cells.

Analysis of melanogenesis protein expression by western blot

B16F10 cells were treated with 10 μM of norartocarpetin and in the presence or absence of α-MSH in a 6-well plate for 48 h. Cells were then collected and lysed in radioimmunoprecipitation assay (RIPA) buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 2 g/ml each of aprotinin, leupeptin, and pepstatin. The lysates were centrifuged at 15,000 rpm for 10 min at 4°C before the supernatant was collected. The protein samples were then denatured and subjected to SDS-PAGE using a 12% running gel, before being transferred onto nitrocellulose membranes. Membranes were incubated with the following primary antibodies for 24 h: p-CREB, MITF, TYR, TRP1, TRP-2, p-ERK, p-p38, p-JNK, or GAPDH, and then incubated with anti-mouse or anti-rabbit horseradish peroxidase antibody for 1 h. The bands of protein expression were developed using ECL reagents and visualized using the Alphatec system. All determinations were performed in triplicate.

Statistical analysis

All data were expressed as mean ± standard deviations of the indicated number of experiments. Statistical significance was determined using Student's t test; a p value of <0.05 was considered significant.

Results

Norartocarpetin is a noncytotoxicity and non-skin irritation compound

To be effective, active whitening compounds should decrease the melanin content in B16F10 melanoma cells with low cytotoxicity. To test the cytotoxicity of norartocarpetin, we treated B16F10 cells with various concentrations of norartocarpetin (5, 10, 20, and 40 μM) and determined cell viability using an MTT assay. As shown in Figure 2A, norartocarpetin concentrations ranging from 5 to 40 μM had no effect on cell viability after 48 h of treatment. In addition, Figure 2B indicated that the same concentration of norartocarpetin did not have any cytotoxic effects on human dermal fibroblasts. Moreover, the skin irritation of active ingredient is the major index of dermal safety in cosmetic application and therefore we conducted the Draize skin irritation test in BALB/c nude mice to confirm the skin irritation of norartocarpetin ( Figure 2C). The three dose of norartocarpetin and vehicle solution were respectively scored 0 (no observable erythema or edema) according to the scoring system of Draize test. Based on the results from skin irritation test, no observable erythema or edema was found on the application site of BALB/c nude mice. The results indicated that norartocarpetin was found to have no skin irritation effect. Together, these results suggest that norartocarpetin might be a noncytotoxic and nonirritation compound for human medical and cosmetic applications. Therefore, we chose norartocarpetin concentrations of 1-10 μM to examine cellular melanin content and tyrosinase assay due to its non-cytotoxicity.

Norartocarpetin effectively decreased cellular melanin content by inhibiting tyrosinase activity

Tyrosinase is a rate-limiting enzyme in melanin biosynthesis, and enhancement of cellular melanin content plays an important role in melanogenesis [19][20][21]. Therefore, a good skin whitening agent would not only effectively inhibit cellular tyrosinase activity but also decrease melanin content. Figure 3A compares melanin content from B16F10 cells treated with DMSO (as control group) compared to those treated with norartocarpetin. Results show that the melanin contents of B16F10 cells treated with 0.01, 0.1, 1, and 10 μM of norartocarpetin have significantly decreased melanin content (P < 0.05), 81.08% ± 3.10%, 79.50% ± 3.89%, 70.13% ± 3.47%, and 50.06% ± 11.94%. Similar results were obtained for cellular tyrosinase activity assay ( Figure 3B). B16F10 cells treated with 0.01, 0.1, 1, and 10 μM of norartocarpetin had significantly lower cellular tyrosinase activity (P < 0.05), i.e., 72.62% ± 6.48%, 73.96% ± 9.68%, 66.24% ± 3.42%, and 55.06% ± 4.81%, respectively. These results indicated that the treatment of B16F10 cells with various concentrations of norartocarpetin not only markedly decreased melanin content but also inhibited tyrosinase activity in a dose-dependent manner.

Norartocarpetin inhibited tyrosinase activity by downregulating MITF and p-CREB protein

It is well known that the synthesis of TYR, TRP-1, and TRP-2 is closely regulated through the activation of MITF and p-CREB protein [22][23][24]. Therefore, we used a western blot assay to determine the effect of various concentrations of norartocarpetin on the protein levels of MITF, p-CREB, TYR, TRP-1, and TRP-2. As shown in Figure 4, p-CREB and MITF are present in control melanoma cells that did not receive norartocarpetin treatment. Tyrosinaserelated proteins (TYR, TRP-1, TRP-2) were also present in B16F10 cells that were not treated with norartocarpetin. These results indicated that B16F10 cells expressed tyrosinase-related proteins through the production of MITF and p-CREB protein. In B16F10 cells treated with norartocarpetin, we observed a dose-dependent decrease in p-CREB and MITF protein levels (P < 0.05). In turn, decreased TYR, TRP-1, and TRP-2 protein levels were also seen. This was particularly clear in the cells treated with 10 μM of norartocarpetin, which had obvious downregulation of p-CREB, MITF, TYR, TRP-1, and TRP-2. These results indicated that norartocarpetin inhibited tyrosinaserelated protein levels, which is known to decrease melanin synthesis.

Norartocarpetin can also inhibit α-MSH-induced melanogenesis α-MSH is usually used to induce MITF protein overproduction, which leads to tyrosinase synthesis and melanin content enhancement, thereby causing melanogenesis. We therefore also treated B16F10 cells with 10 μM of norartocarpetin in an α-MSH-induced melanogenesis assay. Figure 5A indicates that α-MSH dramatically increased melanin content (145.83% ± 0.86%) when compared with the control. We found that treatment with 10 μM of norartocarpetin effectively decreased the melanin content (99.82% ± 2.07%) in α-MSH-induced B16F10 cells (P < 0.05). In addition, Figure 5B shows that 10 μM tyrosinase proteins, and decreases melanin synthesis [25,26]. Therefore, we determined the effects of 10 μM of norartocarpetin on the levels of p-ERK, p-JNK, and p-p38 in a time course experiment. As shown in Figure 6, 10 μM of norartocarpetin enhanced ERK kinase, p38 kinase, and JNK kinase phosphorylation at 3, 6, and 1 h, respectively (P < 0.05). These data indicated that norartocarpetin may induce phosphorylation of three MAPKs and therefore,  Figure 7, inhibition of p38 and JNK MAPKs by their selective inhibitors significantly reversed the antimelanogenesis activity of 10 μM of norartocarpetin (P < 0.05); however, there was no significant reverse effect on ERK inhibition. These results suggest that the antimelanogenesis activity of norartocarpetin depends on phosphorylation of the p38 and JNK pathways but not the ERK pathway.

Discussion

In years past, hydroquinone, a skin whitening agent, is one of the most effective inhibitors of melanogenesis in vitro and in vivo; however, due to cytotoxic effects on melanocytes, it has a side effect of hypopigmentation, which can lead to vitiligo [26,27]. In addition, another common side effect of hydroquinone is skin peeling, redness, or skin sting. Based on these side effects, hydroquinone can not add into cosmetic for preventing skin darkness. Therefore, safety assessment is the first and major consideration in developing drug, health food and cosmetic. In cosmetic industry, the evaluation of cytotoxicity in vitro and skin irritation in vivo of active ingredient is the major index of dermal safety before drug and/or cosmetic product application. Many reports have recently indicated that skin whitening compounds shall be possessed non-cytotoxic effect for determining antimelanogenesis, such as quercetin [28], chrysin [29]. The present study performed cytotoxicity assays on B16F10 melanoma cells and normal human dermal fibroblasts to determine the cell viability of norartocarpetin. Our results demonstrated that norartocarpetin (5-40 μM) did not show significant cytotoxicity towards B16F10 cells (Figure 2A) or normal human dermal fibroblasts ( Figure 2B). In addition, the dermal safety of active ingredient is the first consideration in cosmetic application, such as skin irritation. Our results demonstrated that norartocarpetin did not observe any erythema and edema in Draize test ( Figure 2C). Based on these results, norartocarpetin is a non-cytotoxic and non-irritation compound and therefore the concentrations of norartocarpetin in the above range are used to determine the cellular melanin content, tyrosinase activity, and the molecular biological mechanism of antimelanogenesis.

There are several c-AMP activators used to induce the melanogenesis for developing skin whitening product, such as 3-Isobutyl-1-methylxanthine and α-MSH. It is well-known that α-MSH is a cAMP activator in human and vertebrate animal and which binds to melanocortin-1 receptor on melanocytes to stimulate the production of melanin and result in melanogenesis [18]. When taken in at stimulate dose, α-MSH becomes a potent melanogenesis agent, overproducing the cellular melanin content and tyrosinase activity. According that, the present study used α-MSH as melanogenesis activator to evaluate the antimelanogenesis activity of norartocarpetin. The results of the present study have demonstrated that 10 μM of norartocarpetin is effective as an antimelanogenesis agent since it decreases melanin content ( Figure 3A) and tyrosinase activity ( Figure 3B) in B16F10 cells. In addition, norartocarpetin can also decrease the α-MSH-activated melanogenesis effect that is usually used to stimulate melanin production in B16F10 cells ( Figure 5A). Taken together, these results suggest that norartocarpetin is an effective tyrosinase inhibitor to decrease the melanin production in normal or α-MSH-stimulated conditions. Moreover, the overexpression of tyrosinase is the major rate-limiting step in melanin production. Many reports have demonstrated that CREB phosphorylation induces MITF protein enhancement, which in turn increases tyrosinase synthesis (TYR, TRP-1, and TRP-2) [19,22,23]. These tyrosinase-related proteins are the ratelimiting enzymes of melanogenesis and increase (1) the conversion of tyrosine to dopaquinone, (2) the rearrangement of DOPAchrome to 5,6-dihydroxy-indole-2-carboxylic acid, and (3) the overproduction and accumulation of melanin pigments in skin. Therefore, skin whitening ingredients such as paeonol [30] and curcumin [31] are effectively downregulated p-CREB and MITF proteins, as well as inhibited tyrosinase synthesis, so as to decrease melanin production. Our results demonstrate that norartocarpetin significantly downregulated the level of p-CREB, MITF, and its related proteins, including TYR, TRP1, and TRP2, in a dose-dependent manner. In addition (Figure 4), our data also demonstrated that α-MSH dramatically induced protein expression of MITF and increased the protein levels of TYR, TRP-1, and TRP-2. Our results also indicated that norartocarpetin treatment could diminish α-MSH-induced MITF protein levels, which resulted in reduced TYR, TRP-1, TRP-2 ( Figure 5). In accordance with these findings, norartocarpetin treatment effectively decreased melanin production in B16F10 cells ( Figure 3A) and/or α-MSHinduced B16F10 melanogenesis ( Figure 5A). On the other hand, previous studies have demonstrated that the MAPK signaling pathways (ERK, JNK, and p38) are major regulators of melanogenesis [20,24,30]. MAPK activation plays an important role in inducing MITF phosphorylation at serine-73, which leads to ubiquitination and subsequent MITF degradation, finally diminishing tyrosinase synthesis and melanin production [32]. Skin whitening agents that activate MAPK phosphorylation have been demonstrated to downregulate MITF protein expression and inhibit tyrosinase-related protein synthesis and melanin production [24,25,[28][29][30]. Our study was firstly revealed that norartocarpetin can cause a significant increase in phosphorylation of ERK, JNK, and p38 MAPKs in a time-dependent manner. Activation of MAPKs downregulated MITF protein expression and further diminished tyrosinase (TRP-1 and TRP-2) synthesis, thereby inhibiting melanogenesis. Moreover, when we examined if the modulation of melanin production by norartocarpetin was regulated by MAPK signaling, we found that pretreatment with SB202190 (a selective inhibitor of p38) and SP600125 (a selective inhibitor of JNK) significantly reversed norartocarpetin-reduced melanin production. However, pretreatment with UO126 (a selective inhibitor of MAPK/ERK) did not reverse this. Thus, norartocarpetindecreased melanin production was mediated through both the JNK and p38 pathways, consistent with reports indicating that activation of MAPKs inhibits melanin production in B16F10 melanoma cells.

On a different note, Alesiani et al. demonstrated that high concentrations of 5,7-dimethoxycoumarin (100-500 μM) showed the in vitro anticancer activity in melanoma cells through cell cycle arrest, differentiation induction and the compound can also inhibit the ERK 1/2 phosphorylation led to the B16 cell differentiation and melanogenesis process [33]. Gismondi et al. are also found that nimesulide, a non-steroidal anti-inflammatory drug, played as an antineoplastic agent to induce B16-F10 melanoma cell differentiation by enhancing the transglutaminase and tyrosinase activity and increase of melanin production [34]. In Norartocarpetin Norartocarpetin Norartocarpetin Antimelanogenesis Figure 7 The antimelanogenesis mechanism of norartocarpetin. Norartocarpetin decreases cellular melanin production and tyrosinase activity by activating the phosphorylation of JNK and p38 and which results in reducing of MITF protein and p-CREB and inhibiting tyrosinase related protein synthesis including tyrosinase TRP-1 and TRP-2.

addition, Chen et al. revealed that α-MSH is a cancer stem cell-associated marker in melanoma through upregulating Wnt-1, β-catenin and MITF expression. Resveratrol at 15 μM could downregulate α-MSH stimulated cancer stem cell-associated molecules (Wnt-1, β-catenin and MITF expression) in melanoma B16 cells and finally decreased the cell proliferation, migration, and differentiation [35]. Moreover, Yajima et al. mentioned that MITF plays a "Two Faced" function role in melanoma development and progression. A low level of MITF expression promotes cell proliferation but a high level enhances cell differentiation through induction of cellular senescence and melanogenesis [36]. In the present data, norartocarpetin can downregulate the MITF expression and inhibit the melanogenesis and therefore it implicated that the anticancer activity of norartocarpetin is similar to resveratrol but the mechanism of norartocarpetin merits further investigation for cancer prevention application.

Conclusion

The present study was firstly demonstrated that norartocarpetin is a safe compound due to noncytotoxicity and non-skin irritation. Norartocarpetin decreases cellular melanin production and tyrosinase activity by activating the phosphorylation of JNK and p38 and which results in reducing of MITF protein and p-CREB and inhibiting tyrosinase related protein synthesis including tyrosinase TRP-1 and TRP-2. Consequently, we suggest that as a result of its inhibitory effects on antimelanogenesis, norartocarpetin could be utilized as a skin whitening agent in the treatment of hyperpigmentation diseases and skin whitening cosmetics.

Figure 1
1Chemical structure of norartocarpetin.

H-NMR (400 MHz, Acetone-d 6 ): δ 6.19 (1H, d, J = 2.4 Hz, H-6), 6.42 (1H, d, J = 2.4 Hz, H-8), 6.42 (1H, d, J = 2.4 Hz, H-3′), 6.60 (1H, dd, J = 8.8, 2.4 Hz, H-5′), 7.15 (1H, s, H-3), 7.79 (1H, d, J = 8.8 Hz, H-6′), 13.14 (1H, s, OH-5); 13 C-NMR (100 MHz, Acetone-d 6 ): δ 94.8 (C-8), 99.8 (C-6), 104.1 (C-3′), 105.1 (C-4a), 108.3, (C-3), 109.1 (C-5′), 110.7 (C-1′), 131.0 (C-6′), 159.4 (C-2′), 160.4 (C-4′), 163.1 (C-5), 163.3 (C-8a), 164.2 (C-7), 165.8 (C-2), 184.4 (C-4). Norartocarpetin was collected and stored in a moisture-proof container until further use.

Figure 2
2The in vitro and in vivo safety of norartocarpetin. Cell viability of norartocarpetin in B16F10 (A) and human dermal fibroblast cells (B) and skin irritation of norartocarpetin in nude mice (C). Data are expressed as mean ± SD of 3 independent experiments. The different superscript letters indicate significant difference at p < 0.05 as analyzed by Student's t test. *Significantly different from control. Scoring system of Draize test: (0): no erythema or no edema, (1): very slight erythema or edema, (2) well-defined erythema or edema, (3): moderate erythema or edema, (4): severe erythema or edema.

Figure 3
3of norartocarpetin effectively decreased the MITF level and inhibited the TYR, TRP-1, and TRP-2 protein levels, which diminished the melanin content of α-MSH-induced B16F10 cells.Norartocarpetin downregulated MITF by activating phosphorylation of MAPKsPrevious studies have demonstrated that phosphorylation of MAPKs effectively degrades MITF, diminishes levels of Norartocarpetin decreases the melanogenesis in B16F10 cells. Cellular melanin (A) and tyrosinase activity (B). Data are expressed as mean ± SD of 3 independent experiments. The different superscript letters indicate significant difference at p < 0.05 as analyzed by Student's t test. *Significantly different from control.

Figure 4
4Norartocarpetin downregulates the tryosinase-related protein expression. (A) p-CREB, (B) MITF, (C) TYR, (D) TRP-1, (E) TRP-2. The different superscript letters indicate significant difference at p < 0.05 as analyzed by Student's t test. *Significantly different from control. change the levels of MITF. The effects norartocarpetin on melanin synthesis were further tested by the addition 10 μM of U0126 (a selective inhibitor of MAPK/ERK), SB202190 (a selective inhibitor of p38), and SP600125 (a selective inhibitor of JNK). As shown in

Figure 5
5Norartocarpetin decreases tyrosinase to inhibit α-MSH-induced melanogenesis. Cellular melanin production (A) and downregulation of tyrosinase-related protein expression (B). Data are expressed as mean ± SD of 3 independent experiments. The different superscript letters indicate significant difference at p < 0.05 as analyzed by Student's t test. *Significantly different from control. #Significantly different from α-MSH-induced melanin production.

Figure 6
6Norartocarpetin inhibits tryosinase expression by activating phospho-MAPK. (A) p-ERK, (B) p-JNK, (C) p-p38. (D) melanin content in cells treated for 24 h with MAPK inhibitors with or without 10 μM of norartocarpetin. The different superscript letters indicate significant difference at p < 0.05 as analyzed by Student's t test. *Significantly different from that at 0 h.

The plant species was authenticated by Dr. Ming-Hong Yen of the Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan. The voucher specimen of A. communis J.R. Forst. & G. Forst (2001-ACHW) has been deposited at the Herbarium of the Department of Fragrance and Cosmetic Science, Kaohsiung Medical University, Kaohsiung, Taiwan. Two kilograms of A. communis heartwood was sliced and immersed in a glass container containing methanol at room temperature. This procedure was repeated 3 times. The methanol extract was blended and concentrated using rotary vacuum evaporation. The
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AcknowledgementsWe thank Tainan district agricultural research and extension station, Council of Agriculture, Taiwan, for the material supplement of A. communis in this study. We sincerely acknowledged the financial support of grants from the National Science Council of Taiwan (99-2313-B-037-001-MY3 and NSC 101-2320-B-037-029) and Chang Gung Medical Research Program Foundation (CMRPF6A0081).Competing interestsThe authors declare that they have no competing interests.
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Norartocarpetin from a folk medicine Artocarpus communis plays a melanogenesis inhibitor without cytotoxicity in B16F10 cell and skin irritation in mice. Ko, BMC Complementary and Alternative Medicine. 13348Cite this article asCite this article as: Ko et al.: Norartocarpetin from a folk medicine Artocarpus communis plays a melanogenesis inhibitor without cytotoxicity in B16F10 cell and skin irritation in mice. BMC Complementary and Alternative Medicine 2013 13:348.