Abstract：
BACKGROUND: Ficus benghalensis, commonly known as Banyan Fig, is the national tree of India and its aerial roots are used traditionally to treat female reproductive disorders. However, despite this traditional use, no pharmacological evidence could be traced supporting this use. Additionally, no comprehensive metabolite profiling was reported for F. benghalensis aerial roots.
OBJECTIVE: This study attempts to justify biochemically the traditional use of F. benghalensis aerial roots in treatment of female reproductive disorders and in relation to its secondary metabolite profile.
METHODS: Total ethanol extract (TEE) and subfractions [petroleum ether (PEF), chloroform (CHF), ethyl acetate (EAF) and n-butanol (BUF] were prepared from air-dried powdered aerial roots of F. benghalensis. Detailed in-vivo investigation of the hormonal activity and action mechanism of the total ethanol extract and subfractions was carried out through evaluation of estrogenic and gonadotropic activities. The estrogenic activity was evaluated on ovariectomized immature female rats through estimating uterine weight, vaginal cornification and serum estradiol level along with histological examination of uteri. The gonadotropic activity was measured by assay of follicle stimulating hormone (FSH) and luteinizing hormone (LH) like activities. Total follicular and corpora lutea counts in immature female rats were used to determine FSH and LH like activities, respectively in addition to histological picture of the genitalia. Comprehensive non-targeted metabolite profiling was carried out for the TEE and subfractions using UPLC-HRMS in negative and positive ionization modes. UPLC-MS fingerprint was subjected to principal component analysis (PCA) and partial least squares analyses to correlate the bioactivities to specific chemical constituents in F. benghalensis different subfractions. GC-MS was further used for non-polar silylated fractions.
RESULTS: Results revealed that only the non-polar PEF and CHF displayed moderate estrogenic and FSH-like activities but with no LH-like activity. Metabolites profiling via (UPLC-HRMS) and multivariate PCA analysis enabled identification and comparison of various chemical classes in F. benghalensis extract and fractions. The active non-polar fractions revealed nearly similar metabolites profile being composed of isoflavonoids, triterpenes, sterols, fatty acids and cyclic peptides. In contrast, polar fractions were more abundant in apocarotenoids, fatty acyl amides, hydroxybenzoates and hydroxycinnamates in addition to two lignans. PLS analysis revealed strong correlation between hydroxylated fatty acids and pyranoisoflavones with estrogenic and FSH-like activities. GC-MS analysis was further employed for non-polar fractions profiling revealing for their enrichment in fatty acids/esters, terpenes, organic acids and phenolics.
CONCLUSIONS: This is the first study to rationalize the use of F. benghalensis aerial root traditionally in treatment of gynecological disorders, revealing that the petroleum ether and chloroform non-polar subfractions of F. benghalensis showed estrogenic and FSH-like activity with absence of LH-like activity. This biological activity could possibly be attributed to its metabolites profile of isoflavonoids, fatty acids, triterpenes, sterols and cyclic peptides identified via UPLC-MS and GC-MS techniques. Consequently, F. benghalensis aerial roots should be used with caution in traditional treatment of female infertility or other reproductive disorders.
OBJECTIVE: This study attempts to justify biochemically the traditional use of F. benghalensis aerial roots in treatment of female reproductive disorders and in relation to its secondary metabolite profile.
METHODS: Total ethanol extract (TEE) and subfractions [petroleum ether (PEF), chloroform (CHF), ethyl acetate (EAF) and n-butanol (BUF] were prepared from air-dried powdered aerial roots of F. benghalensis. Detailed in-vivo investigation of the hormonal activity and action mechanism of the total ethanol extract and subfractions was carried out through evaluation of estrogenic and gonadotropic activities. The estrogenic activity was evaluated on ovariectomized immature female rats through estimating uterine weight, vaginal cornification and serum estradiol level along with histological examination of uteri. The gonadotropic activity was measured by assay of follicle stimulating hormone (FSH) and luteinizing hormone (LH) like activities. Total follicular and corpora lutea counts in immature female rats were used to determine FSH and LH like activities, respectively in addition to histological picture of the genitalia. Comprehensive non-targeted metabolite profiling was carried out for the TEE and subfractions using UPLC-HRMS in negative and positive ionization modes. UPLC-MS fingerprint was subjected to principal component analysis (PCA) and partial least squares analyses to correlate the bioactivities to specific chemical constituents in F. benghalensis different subfractions. GC-MS was further used for non-polar silylated fractions.
RESULTS: Results revealed that only the non-polar PEF and CHF displayed moderate estrogenic and FSH-like activities but with no LH-like activity. Metabolites profiling via (UPLC-HRMS) and multivariate PCA analysis enabled identification and comparison of various chemical classes in F. benghalensis extract and fractions. The active non-polar fractions revealed nearly similar metabolites profile being composed of isoflavonoids, triterpenes, sterols, fatty acids and cyclic peptides. In contrast, polar fractions were more abundant in apocarotenoids, fatty acyl amides, hydroxybenzoates and hydroxycinnamates in addition to two lignans. PLS analysis revealed strong correlation between hydroxylated fatty acids and pyranoisoflavones with estrogenic and FSH-like activities. GC-MS analysis was further employed for non-polar fractions profiling revealing for their enrichment in fatty acids/esters, terpenes, organic acids and phenolics.
CONCLUSIONS: This is the first study to rationalize the use of F. benghalensis aerial root traditionally in treatment of gynecological disorders, revealing that the petroleum ether and chloroform non-polar subfractions of F. benghalensis showed estrogenic and FSH-like activity with absence of LH-like activity. This biological activity could possibly be attributed to its metabolites profile of isoflavonoids, fatty acids, triterpenes, sterols and cyclic peptides identified via UPLC-MS and GC-MS techniques. Consequently, F. benghalensis aerial roots should be used with caution in traditional treatment of female infertility or other reproductive disorders.
摘要：
背景：本加榕树，俗称榕树无花果，是印度的国树，其气根传统上用于治疗女性生殖障碍。然而,尽管有这种传统的用法,没有药理学证据支持这种使用。此外,没有关于F.benghalensis气根的全面代谢物分析的报道。
目的：本研究试图从生物化学上证明本加根草在治疗女性生殖疾病中的传统使用及其次生代谢产物谱。
方法：总乙醇提取物（TEE）和亚组分[石油醚（PEF），氯仿(CHF),乙酸乙酯（EAF）和正丁醇（BUF）是从风干的F.benghalensis的气生根中制备的。通过评估雌激素和促性腺激素活性，对总乙醇提取物和亚组分的激素活性和作用机理进行了详细的体内研究。通过估计子宫重量来评估卵巢切除的未成熟雌性大鼠的雌激素活性，阴道角质化和血清雌二醇水平以及子宫的组织学检查。促性腺激素活性通过卵泡刺激素（FSH）和黄体生成素（LH）样活性测定来测量。未成熟雌性大鼠的总卵泡和黄体计数用于确定FSH和LH样活动，除了生殖器的组织学图片。使用UPLC-HRMS以负和正电离模式对TEE和亚组分进行全面的非靶向代谢物分析。对UPLC-MS指纹进行主成分分析（PCA）和偏最小二乘法分析，以将生物活性与本加不同亚组分中的特定化学成分相关联。GC-MS进一步用于非极性甲硅烷基化级分。
结果：结果显示，只有非极性PEF和CHF表现出中等的雌激素和FSH样活性，但没有LH样活性。通过（UPLC-HRMS）和多变量PCA分析进行代谢物分析，可以识别和比较本加尔氏弧菌提取物和馏分中的各种化学类别。活性非极性部分显示出几乎相似的代谢物分布由异黄酮组成，三萜,固醇,脂肪酸和环肽。相比之下,极性部分在载脂蛋白类胡萝卜素中更丰富，脂肪酰酰胺，除了两种木酚素外，还有羟基苯甲酸酯和羟基肉桂酸酯。PLS分析显示，羟基化脂肪酸和吡喃异黄酮与雌激素和FSH样活性之间存在很强的相关性。进一步采用GC-MS分析进行非极性级分分析，揭示其在脂肪酸/酯中的富集，萜烯,有机酸和酚类。
结论：这是第一个在传统上合理使用F.benghalensis气根治疗妇科疾病的研究，揭示了F.benghalensis的石油醚和氯仿非极性亚组分显示出雌激素和FSH样活性，而没有LH样活性。这种生物活性可能归因于其异黄酮的代谢物谱，脂肪酸，三萜,通过UPLC-MS和GC-MS技术鉴定的甾醇和环肽。因此,在女性不育或其他生殖障碍的传统治疗中，应谨慎使用F.benghalensis气根。
目的：本研究试图从生物化学上证明本加根草在治疗女性生殖疾病中的传统使用及其次生代谢产物谱。
方法：总乙醇提取物（TEE）和亚组分[石油醚（PEF），氯仿(CHF),乙酸乙酯（EAF）和正丁醇（BUF）是从风干的F.benghalensis的气生根中制备的。通过评估雌激素和促性腺激素活性，对总乙醇提取物和亚组分的激素活性和作用机理进行了详细的体内研究。通过估计子宫重量来评估卵巢切除的未成熟雌性大鼠的雌激素活性，阴道角质化和血清雌二醇水平以及子宫的组织学检查。促性腺激素活性通过卵泡刺激素（FSH）和黄体生成素（LH）样活性测定来测量。未成熟雌性大鼠的总卵泡和黄体计数用于确定FSH和LH样活动，除了生殖器的组织学图片。使用UPLC-HRMS以负和正电离模式对TEE和亚组分进行全面的非靶向代谢物分析。对UPLC-MS指纹进行主成分分析（PCA）和偏最小二乘法分析，以将生物活性与本加不同亚组分中的特定化学成分相关联。GC-MS进一步用于非极性甲硅烷基化级分。
结果：结果显示，只有非极性PEF和CHF表现出中等的雌激素和FSH样活性，但没有LH样活性。通过（UPLC-HRMS）和多变量PCA分析进行代谢物分析，可以识别和比较本加尔氏弧菌提取物和馏分中的各种化学类别。活性非极性部分显示出几乎相似的代谢物分布由异黄酮组成，三萜,固醇,脂肪酸和环肽。相比之下,极性部分在载脂蛋白类胡萝卜素中更丰富，脂肪酰酰胺，除了两种木酚素外，还有羟基苯甲酸酯和羟基肉桂酸酯。PLS分析显示，羟基化脂肪酸和吡喃异黄酮与雌激素和FSH样活性之间存在很强的相关性。进一步采用GC-MS分析进行非极性级分分析，揭示其在脂肪酸/酯中的富集，萜烯,有机酸和酚类。
结论：这是第一个在传统上合理使用F.benghalensis气根治疗妇科疾病的研究，揭示了F.benghalensis的石油醚和氯仿非极性亚组分显示出雌激素和FSH样活性，而没有LH样活性。这种生物活性可能归因于其异黄酮的代谢物谱，脂肪酸，三萜,通过UPLC-MS和GC-MS技术鉴定的甾醇和环肽。因此,在女性不育或其他生殖障碍的传统治疗中，应谨慎使用F.benghalensis气根。
