{Studies on the chemical compositions of Hyptis suaveolens (L.) poit} J. Serb. Chem. Soc. 84 (3) 245–252 (2019) UDC 582.94:581.19:544.2.004.12:547(510) JSCS–5180 Original scientific paper 245 Studies on the chemical compositions of Hyptis suaveolens (L.) Poit. GENGQIU TANG, XILE LIU, XUE GONG, XIAOJING LIN, XIUDI LAI, DONG WANG and SHENGGUO JI* School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, P. R. China (Received 8 December 2017, revised 21 May, accepted 20 September 2018) Abstract: This study aimed at identifying the chemical compounds isolated from the aerial parts and roots of Hyptis suaveolens (L.) Poit. The compounds were isolated and purified by a combination of various chromatographic techniques including silica gel column chromatography, silica gel reverse phase column chromatography, Sephadex LH-20 gel column chromatography, semi-preparative HPLC and recrystallization. The chemical structures were analyzed and elucidated based on physiochemical properties and spectroscopic analysis. From the aerial parts of H. suaveolens, eight compounds were isolated and identified as quercetin 3-O-β-D-glucopyranoside (1), apigenin (2), sorbi- folin (3), quercetin (4), kaempferol (5), genkwanin (6), rosmarinic acid (7) and methyl rosmarinate (8). Two compounds were isolated from the roots of H. suaveolens and identified as podophyllotoxin (9) and picropodophyllotoxin (10). Compounds 2–6 were isolated from H. suaveolens for the first time while compound 10 was isolated from the genus of Hyptis for the first time. The results of this study provided a scientific basis for the development of the medi- cinal value of H. suaveolens and have important theoretical significance for the chemical utilization of H. suaveolens resources. Keywords: structural identification; quercetin 3-O-β-D-glucopyranoside; sorbifolin. INTRODUCTION Hyptis suaveolens (L.) Poit, locally known in China as Maolaohu belongs to the family Lamiaceae, covers important horticultural crops having economical value and is an important medicinal plant that is distributed in dense clumps along roadsides, in over-grazed pastures and around stockyards throughout the tropics and subtropics. H. suaveolens has a number of synonyms, such as Ballota suave- olens L., Bystropogon graveolens Blume, Bystropogon suaveolens (L.) L'Hér., Gnoteris cordata Raf., Gnoteris villosa Raf., Hyptis congesta Leonard, Hyptis * Corresponding author: E-mail shengguo_ji@163.com https://doi.org/10.2298/JSC171208078T 246 TANG et al. ebracteata R.Br., Hyptis graveolens Schrank, Hyptis plumieri Poit., Marrubium indicum Blanco, Mesosphaerum suaveolens (L.) Kuntze, Schaueria graveolens (Blume) Hassk. or Schaueria suaveolens (L.) Hassk. H. suaveolens is a fast-grow- ing perennial and aromatic herb, 0.4–2 m tall with hairy stems and leaves, having branches and usually woody at the base. The leaves are weak, oval in outline, tip broadly pointed.1,2 The plant is native to the tropics of America and now consi- dered as a weed worldwide. There are also many distributions on the southern coast of China. The medicinal use of the plant has been reported in 22 countries, which emphasizes its potential values of application in modern pharmacy. In trad- itional medicine, almost all parts of this plant are used to treat numerous ailments, such as respiratory and gastrointestinal infections, as antirheumatic and antisup- orific baths, to treat indigestion, colic, stomachache, colds, fever, burns, wounds, cramps and various skin complaints.3–6 H. suaveolens has both medicinal indi- viduality as well as insecticidal properties. In several countries, it is commonly used as an insect repellent by incinerating or placing it naturally.7–10 Among these traditional medicinal uses, the leaves are used most frequently, followed by seeds and thirdly the whole plants. In addition, the decoction of the roots was reported to contain urosolic acid, a natural HIV integrase inhibitor, and was highlighted for the preparation of an appetizer.11 The chemical components of the essential oils obtained from the leaves of H. suaveolens were intensively investigated in previous studies. Many new chemical compounds, such as monoterpenes and sesquiterpen- oids, obtained from the essential oils of H. suaveolens leaves were isolated and their structures elucidated, and their antibacterial, antifungal and anti-Aspergillus activity confirmed.12–16 In addition, it was reported that the leaves had anticancer and antifertility properties, while their aqueous extract showed an antinociceptive effect and acute toxicity, especially for the essential oil.17,18 However, little attention has been given to the extraction, separation and structural identification of chemical compositions from the entire plant, leading to a lack of a systematic study. Therefore, information concerning the chemical composition of H. suave- olens is still incomplete. As the drug is endowed with huge exploitation and util- ization value, it is medicinally important to know precisely and comprehensively its chemical composition. In view of the importance of this, the present investig- ation is concerned with the extraction, separation, purification and structural iden- tification of the aerial parts and roots of H. suaveolens using physicochemical and chromatographic methods to help in the development of the utilization and phar- macological activity of this drug. MATERIALS AND METHODS General procedures All reagents used for the analyses were of analytical grade and were not subjected to further purification. Column chromatography was performed on silica gel (200–300 mesh, Qingdao Haiyang Chemical Works, China), ODS (50–70 µm, Tianjin Jianhexiang Chromato- CHEMICAL COMPOSITIONS OF Hyptis suaveolens (L.) 247 graphic Works, China) and Sephadex LH-20 (Beijing Ruida Henghui Scientific and Technical Corporation, China). Semi-preparative HPLC was realized on a Gilson 322 instrument, using a Shim-pack GIS C18 column (3.0 cm×250 mm, 5 µm, Shimadzu, Japan). The ESI-MS spec- tra were recorded on a Waters ACQUITY UPLC/Q-TOF micro LC-MS spectrometer (Waters, USA). The NMR spectra were obtained on an AVANCE 500 spectrometer (Bruker, Germany). Collection, identification and preparation of the plant materials Fresh whole plants of H. suaveolens growing on the grounds of Guangdong Pharma- ceutical University, Guangzhou Higher Education Mega Center were randomly collected, taxonomically identified and authenticated by Prof. Shengguo Ji, School of Traditional Chin- ese Medicine, Guangdong Pharmaceutical University. They were cleaned to remove physical impurities, air-dried in the shade, mechanically powdered, sieved using 10 meshes and stored in hermetic containers under dry air for further studies. This research was conducted in the Key Laboratory of the State Administration of Traditional Chinese Medicine for the Product- ion & Development of Cantonese Medicinal Materials, School of Traditional Chinese Medi- cine, Guangdong Pharmaceutical University, China, during 2016–2017. Extraction and purification of samples The coarse powder materials of aerial parts (8.67 kg) and roots (1 kg) of H. suaveolens were subjected to ultrasonic extraction three times with ethanol (90 % and 2×70 %, the ratio of solid (powder) to liquid (ethanol) was 1:10), and these extracts were concentrated in a rot- ary evaporator under reduced pressure and controlled temperature (< 45 °C). They were then extracted separately and successively with petroleum ether, ethyl acetate and n-butanol. (b.p. 60–90 °C, 10 L). The ethyl acetate extracts were placed in an airtight container and stored in a refrigerator. Purification of the samples were performed using silica gel column chromato- graphy, silica gel reverse phase column chromatography, Sephadex LH-20 gel column chro- matography, Gilson semi-preparative chromatography and recrystallization. Identification of constituents The constituents of the extracts were determined by a combination of physicochemical properties, UV spectrometry and nuclear magnetic resonance spectrometry (NMR), and iden- tified by comparing their mass spectral patterns with the data for original samples and by matching those cited in the literature.19-28 RESULTS AND DISCUSSION The chemical compounds of the aerial parts and roots of H. suaveolens were determined and are listed in Table I. Their structures were identified by com- parison of physicochemical analysis and their spectral data (ESI-MS, 1H-NMR and 13C-NMR data) with literature values. The characteristics, solubility and melting points (m.p.) of compounds are also given in Table I, which were used to deduce the functional group. By comparing with the UV absorption and ESI-MS data, given in Table II, the molecular formula of each compound could be obtained. The 13C-NMR and 1H-NMR spectra data are presented in Tables S-I and S-II, respectively, of the Supplementary material to this paper. From these data, det- ailed structural information of compounds, shown in Fig. 1, were obtained. The results revealed that the identified compounds from the aerial parts of H. suaveolens included quercetin 3-O-β-D-glucopyranoside (1), apigenin (2), sorbi- 248 TANG et al. folin (3), quercetin (4), kaempferol (5), genkwanin (6), rosmarinic acid (7) and methyl rosmarinate (8), while the identified compounds from the roots of this plant included podophyllotoxin (9) and picropodophyllotoxin (10). Compounds 2–6 were found in H. suaveolens for the first time, while compound 10 was iso- lated for the first time from the genus of Hyptis. Compound 1, also called iso- quercetin, was found in this plant in a previous study,29 which was identified in course of LC-MS measurements, but it was not isolated. Compounds 7–9 were also obtained from the LC-MS in this plant according to previous literature.30,31 TABLE I. Components detected in the extracts of H. suaveolens with solubility and melting point data Compound Characteristics Solubility M.p. / °C Functional group classes Quercetin 3-O-β-D-glu- copyranoside Yellow powder Methanol-soluble; water- and ethyl acetate-insoluble – Flavonoid glycoside Apigenin Yellow powder Methanol-soluble; water-insoluble 347–348 Flavone Sorbifolin Yellowish white needle crystals Methanol-soluble; water-insoluble n.a. Flavone Quercetin Yellow powder Methanol-microsoluble; Chloroform and ethyl acetate-insoluble 313–314 Flavone Kaempferol Yellow powder Methanol-microsoluble; chloroform- and ethyl acetate-insoluble 276–278 Flavone Genkwanin Yellow powder Methanol- and water-insoluble 293–296 Flavone Rosmarinic acid White powder Water- and methanol- -soluble; acetone-insoluble 171–175 Polyphenol Methyl rosmarinate Yellow syrup Methanol- and ethyl acetate-soluble; water-insoluble – Polyphenol Podophyllotoxin White powder Methanol-soluble; water-insoluble 183–184 Lignan Picropodophyllotoxin White powder Acetone-soluble; water-insoluble 225–227 Lignan TABLE II. UV and ESI-MS data for the isolated compounds Compound Max. absorption in UV, nm ESI-MS Exact mass Molecular formula Ref. Quercetin 3-O-β-D-glu- copyranoside 268, 364 487.0857[M+Na]+, 463.0887[M–H]－ 465.391 C21H20O12 19 Apigenin 268, 336 271.0635[M+H]+ 270.241 C15H10O5 20 Sorbifolin 262, 325 323.0542[M+Na]+, 299.0536[M–H]－ 300.268 C16H12O6 21 Quercetin 283, 336 303.0445[M+H]+, 301.0344[M–H]－ 302.201 C15H10O7 22 Kaempferol 222, 267, 366 285.0378[M–H]－ 286.241 C15H10O6 23 Genkwanin – 283.0619[M–H]－ 284.268 C16H12O5 24 CHEMICAL COMPOSITIONS OF Hyptis suaveolens (L.) 249 TABLE II. Continued Compound Max. absorption in UV, nm ESI-MS Exact mass Molecular formula Ref. Rosmarinic acid 199, 261, 325 361.0931[M+H]+, 383.0746[M+Na]+, 359.0785[M–H]－ 360.32 C18H16O8 25 Methyl rosmarinate 205, 264, 331 374.0961[M+H]+, 397.0901[M+Na]+, 373.0933[M–H]－ 374.347 C19H18O8 26 Podophyllotoxin 203, 288 432.0534[M+NH4]+, 415.0847 [M+H]+ 414.412 C22H22O8 27 Picropodophyllotoxin 264, 322 851.0301[2M+Na]+, 438.0634[M+Na+1], 415.0688[M+1]+ 414.412 C22H22O8 28 Fig 1. Chemical structures of 1–10. 1. Quercetin 3-O-β-D-glucopyranoside; 2. apigenin; 3. sorbifolin; 4. quercetin; 5. kaempferol; 6. genkwanin; 7. rosmarinic acid; 8. methyl rosmarinate; 9. podophyllotoxin; 10. picropodophyllotoxin Most previous studies focused on the identification of polyphenols, sesqui- terpenes, diterpenes, triterpenes and steroids extracted from H. suaveolens, but little attention was given to the flavonoids extracted from this plant. It was indi- 250 TANG et al. cated that flavonoids have antibacterial and antiviral, antitumor, antioxidant, anti- inflammatory, analgesic and other biological activities. The results showed that compounds obtained from the aerial parts of H. suaveolens were flavonoid constituents mostly. Generally, dissociative flavonoids were extracted by ethanol (90–95 %), flavonoid glycosides were extracted by ethanol (60 %), while both could be extracted by ethanol (70 %). In this study, the flavonoid constituents of the aerial parts of H. suaveolens were studied by the ultrasonic extraction method using ethanol (70 and 90 %), which could be used for the comprehensive extract- ion of flavonoids. However, in this experiment, tailing of peaks of preparation liquid chromatogram could easily occur when extracting flavonoids monomer. In this case, the hydrothermal re-crystallization purification method can be tried to remove water-soluble impurities and improve the purity of the product when the percentage of methanol in methanol-water is more than 30 %. The compounds podophyllotoxin and picropodophyllotoxin obtained in this research belong to the functional group of aryl lignans that are mainly derived from plants of the family Berberidaceae, and are isomers to each other. It was reported that podophyllo- toxin had potent inhibitory effect on tumors and virus, which reveal the huge exploitation and utilization value of the roots of H. suaveolens.31 This research provides essential information for further studies on the medi- cinal values of H. suaveolens. However, H. suaveolens has not been recorded in the Chinese Pharmacopoeia. In order to develop the curative value of it, a series of further scientific research, such as its pharmacological research, clinical tests and formulation of the quality standard have to be completed. CONCLUSIONS The chemical compounds isolated from the aerial parts and roots of Hyptis suaveolens (L.) Poit. were identified by comparison of physico-chemical analysis and their spectral data. The components obtained from the aerial parts were iden- tified as quercetin 3-O-β-D-glucopyranoside (1), apigenin (2), sorbifolin (3), quercetin (4), kaempferol (5), genkwanin (6), rosmarinic acid (7) and methyl ros- marinate (8), while the components obtained from the roots were identified as podophyllotoxin (9) and picropodophyllotoxin (10). In this regard, compounds 129 and 7–930,31 have been reported to be found in H. suaveolens, while compounds 2–6 are found in H. suaveolens for the first time and compound 10 is isolated from the genus of Hyptis for the first time. SUPPLEMENTARY MATERIAL The 1H-NMR and 13C-NMR data, as well as all recorded spectra and preparation liquid chromatograms, are available electronically at the pages of journal website: http:// //www.shd.org.rs/JSCS/, or from the corresponding author on request. CHEMICAL COMPOSITIONS OF Hyptis suaveolens (L.) 251 И З В О Д ИСПИТИВАЊЕ ХЕМИЈСКОГ САСТАВА Hyptis suaveolens (L.) Poit. GENGQIU TANG, XILE LIU, XUE GONG, XIAOJING LIN, XIUDI LAI, DONG WANG и SHENGGUO JI School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, P. R. China Циљ истраживања је био да се изврши идентификација хемијских једињења која су изолована из надземних делова и корена биљке Hyptis suaveolens (L.) Poit. Једињења су изолована и пречишћена комбиновањем различитих хроматографских и препаративних техника, којe укључују хроматографију на колони силика-гела, хроматографију на реверсној фази модификованог силика-гела, хроматографију на колони Sephadex LH-20, семи-препаративну HPLC хроматографију и кристализацију. Хемијске структуре једињења анализиране су и утврђене на основу физичко–хемијских особина и спектро- скопске анализе. Осам једињења је изоловано из надземних делова H. suaveolens и идентификована су као кверцетин-3-O-β-D-глукопиранозид (1), апигенин (2), сорбифо- лин (3), кверцетин (4), камферол (5), генкванин (6), рузмаринска киселина (7) и метил- -розмаринат (8). Два једињења су изолована из корена H. suaveolens и идентификована су као подофилотоксин (9) и пикроподофилотоксин (10). Једињења 2–6 изолована су по први пут из H. suaveolens док је једињење 10 изоловано из рода Hyptis по први пут. Резултати истраживања пружају научну заснованост за резвијање примене H. suaveolens у медицини и имају важан теоретски значај за ширу употребу H. suaveolens. (Примљено 8. децембра 2017, ревидирано 21. маја, прихваћено 20. септембра 2018) REFERENCES 1. M. R. Henderson, Malayan Wild Flowers (Dicotyledones), Tien Wah Press Ltd., Singapore, 1959, p. 476 2. C. A. Backer, R. C. B. Van de Brink, Jr., Flora of Java, N.V.P. Noordhoff, Graningen, 1965, p. 630 3. K. R. Kiritikar, B. D. 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