# Epifriedelanol and Dammara-20,24-dien-3-yl Acetate from EthiopianInula confertiflora Root Extract

Authors: Minbale Gashu
Journal: Journal of Chemistry
 (2022)

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## Abstract

Phytochemical investigation of the ethanol extract of the root ofInula confertiflora afforded two rare triterpenoids, namely, epifriedelanol and dammara-20,24-dien-3-yl acetate, and their chemical structure was elucidated using appropriate techniques.


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## Body

## 1. Introduction

The herbInula confertiflora (A. Rich, Asteraceae, called Weynagift in Amh.) is an endemic medicinal plant found in Ethiopia. It is locally applicable to treat skin diseases caused by viruses, wounds, and eczematous lesions [1]. Traditionally dried roots of I. confertiflora are smoked as a fumigant during child birth and used in treating leprosy. Maceration of pounded leaves in water is also applied to diseased eyes of cattle and treating asthma and cough [2].Similarly, the roots of I. racemosa, I. helenium, and I. viscosa are reported for their medicinal use by the indigenous people of their origin and are highly studied for their phytochemical profile. The root of I. racemosa is used for handling asthma-like conditions, reducing cholesterol, supporting healthy circulation, and ensuring healthy heart functions. It is also applicable to the management of diabetes. In Chile, I. helenium treats wounds and dandruff, bronchitis, and asthma. In Hungarian traditional medicine, the roots of I. helenium are used against asthma, cough, bronchitis, lung disorders, tuberculosis, and infectious and helminthic diseases. The root powders of I. viscosa are used against cough, hypertension, and diabetes mellitus. Decoction of I. viscosa roots was also used in the treatment of skin irritations of allergic origin in Italian traditional medicine [3, 4].Chemical constituents of several Inula species known elsewhere were well explored, more than 400 compounds were reported, and they are categorized as terpenoids, flavonoids, and glycosides, etc. Of all the isolated compounds, sesquiterpene lactones were the major constituents [5–7]. They are grouped under eudesmanolides, guaianolides, pseudoguianolides, germacranolides, and xanthanolides [8]. These sesquiterpene lactones showed a wide range of biological properties including anticancer, anti-inflammatory, antifungal, and antibacterial activities [5–9]. In this study, the claims of the traditional healers, production of variety of compounds by other Inula species, plant endemicity, and the absence of reported work on phytochemical studies of I. confertiflora root were of interest.


## 2. Experimental

### 2.1. General

All chemicals and solvents used were of analytical grade. Melting point was determined using Thomas Hoover capillary melting point apparatus. TLC was performed on 0.25 mm thick layer of precoated silica gel GF254 (Merck) hexane/ethyl acetate as eluent with vanillin-sulfuric acid as detecting reagent. Column chromatography was performed on silica gel (Merck). NMR spectral measurements were done on Bruker ACQ 400 AVANCE spectrometer operating at 400 MHz. The IR spectra were recorded using a Perkin-Elmer BX Spectrometer (400–4000 cm−1) in KBr. LC-MS/EIMS was done at Korea Research Institute of Chemical Technology.


## 2.1. General

All chemicals and solvents used were of analytical grade. Melting point was determined using Thomas Hoover capillary melting point apparatus. TLC was performed on 0.25 mm thick layer of precoated silica gel GF254 (Merck) hexane/ethyl acetate as eluent with vanillin-sulfuric acid as detecting reagent. Column chromatography was performed on silica gel (Merck). NMR spectral measurements were done on Bruker ACQ 400 AVANCE spectrometer operating at 400 MHz. The IR spectra were recorded using a Perkin-Elmer BX Spectrometer (400–4000 cm−1) in KBr. LC-MS/EIMS was done at Korea Research Institute of Chemical Technology.


## 3. Plant Material and Extraction Method

Root ofI. confertiflora (Figure 1) was collected from Ankober Palace Lodge, identification was made by a professional botanist, and voucher specimen (S1152) of the plant was deposited at the National Herbarium of Ethiopia (AAU). Shade dried roots were ground and placed in brown bottles until used. The plant material (100 g) was macerated using ethanol (95%) with occasional shaking for 72 h, and the solvent was removed under vacuum using rotary evaporator. The crude extract was subjected to phytochemical study.Figure 1
Inula confertiflora leaf (left) and root (right) parts (photo by MG).


## 4. Separation, Purification, and Structure Elucidation of Compounds

Separation of the gummy crude extract of plant material into several fractions and purification of the fractions into different compounds were done by using combination of chromatographic techniques (TLC, CC). The structures of purified compounds were elucidated using the data generated by appropriate spectroscopic methods. The ethanol crude extract of the root (4 g) subjected to CC separation using hexane and ethyl acetate of increasing polarity afforded nine pooled fractions. Epifriedelanol (96–39H, 5 mg) was recrystallized from Fr2 in EtOAc. Repeated chromatographic analysis of the mixture of Fr1 and Fr 3-4 in hex:EtOAc as mobile phase afforded fourteen fractions of which dammara-20, 24-dien-3-yl acetate (96–56A, 25 mg) was recrystallized from Fr1 in EtOAc. The structures of the isolated compounds were elucidated using appropraite spectroscopic techniques such as 1H and 13C-NMR and IR and MS and compared with data in the reported literature.


## 5. Results and Discussion

Phytochemical studies on the ethanol crude extract of the root revealed the presence of epifriedelanol (1) and dammara-20,24-dien-3-yl acetate (2). The structures of these triterpenoid compounds were elucidated by using spectroscopic data and through comparison with facts reported in the literature as follows.Compound1 was obtained as colorless solid (Rf0.46 in hex: EtOAc (1 : 1)) from the root of I. confertiflora. The IR spectrum showed characteristic absorption bands at 3490 cm−1(O-H), 2925/2870 cm−1 (C-H). Its 1H NMR spectrum contained only one downfield doublet at δ3.75 (1H, J = 2.8 Hz) indicating the presence of a proton on an oxygenated carbon (H-3). Although the proton spectrum is complex, there is a proton observed at δ1.92 (1H dt, J = 9.2, 2.8 Hz, H-2a) showing its correlation with H-3. The spectrum also showed doublet at δ 0.90 (3H, d, J = 6.8 Hz, H-23) due to methyl protons attached to a tertiary carbon (C-4) which is related with multiplet signal at δ1.76 (H-4). The seven singlets at δ 0.87, 0.91, 0.94, 0.95, 0.97, 1.01, and 1.18 are attributed to methyl attached to quaternary carbons. The 13C-NMR spectrum showed 30 carbon resonances due to six quaternary, five methine, eleven methylene, and eight methyl groups. The presence of a hydroxyl group inferred from IR spectrum was evident in the13C NMR spectrum from the appearance of an oxymethine (C-3) carbon signal at δ72.8. The 1H and 13C-NMR spectral data indicate that the compound belongs to the friedelane group of triterpenes. It was identified as epifriedelanol by comparing the spectroscopic data with those reported in the literature [10–12] (Table 1).Table 1
13C-NMR data of compound 1 and the data reported in the literature for epifriedelanol (CDCl3,δ in ppm).
C noExp resultsδCLit resultsδC115.815.80235.235.18372.872.78449.149.16537.137.11641.741.72717.517.56853.253.20938.438.381061.361.341135.335.351230.630.651337.837.841439.739.681532.332.331636.136.081730.030.041842.842.811935.535.562028.228.202132.832.812239.339.302311.611.662416.416.422518.218.272618.718.692720.120.152831.831.812935.035.063032.132.11The EIMS also showed a molecular ion peak at m/z 428 corresponding to the molecular formula C30H52O in agreement with compound 1.Compound2 was isolated as white crystals (Rf 0.44 in EtOAc : Hex (1 : 1) from extract of I. confertiflora root. Its molecular formula was determined as C32H52O2 using EIMS analysis (m/z 468 (M+.)). The IR spectrum exhibited absorption bands at 1728 cm−1(C=O) and 2963/2850 cm−1 (C-H). The 1H NMR spectrum (CDCL3) showed the presence of olefinic methylene proton signals (H-21) at δ 4.72 (1H, s) and 4.76 (1H, s), vinyl proton (H-24) at δ5.16 (1H, m), and oxymethine proton (H-3) at δ4.50 (1H,m). Two singlets at δ1.63 (H-26) and 1.71 (H-27) due to allylic methyl groups were also observed. Six additional singlets at δ1.31, 1.27,0.89, 0.87, 0.99, and 2.05 corresponding to H-18, H-19, H-28, H-29, H-30, and acetyl methyl protons were also evident, respectively. The 13C-NMR spectrum (CDCl3) showed thirty-two resolved carbon signals and DEPT-135 spectrum identified them as eight methyl, seven quaternary, eleven methylene, and six methine carbon signals. In the 13C-NMR spectrum, seven singlets at δ16.3 (C-18), 15.7 (C-19), 25.7 (C-26), 17.7 (C-27), 28.0 (C-28), 15.9(C-29), and 16.5 (C-30) were due to methyl groups.The13C-NMR spectrum also showed the presence of one terminal double bond resonating at δ152.7 (C-20) and 107.5 (C-21) and a substituted olefin at δ124.5 (C-24) and 131.4(C-25). Downfield oxymethine carbon signal (δ80.9) along with α-methyl (δ21.3) and carbonyl (δ171.0) signals showed the existence of acetyl group. The overall spectroscopic data were in close agreement with the reported values of dammara-20,24-dien-3-yl acetate (2) [12, 13] (Table 2).Table 2
13C-NMR data of compound 2 and the data reported for dammara-20,24-dien-3-yl acetate (CDCl3,δ in ppm).
C noExp resultsδCLit resultsδC138.837.9223.723.7380.980.9437.938.8556.056.0618.218.2735.435.4840.540.5950.950.91037.237.21121.421.41224.925.01345.345.31449.449.51531.431.41627.127.11747.847.91816.316.31915.715.720152.7152.721107.5107.52234.234.22328.928.924124.5124.525131.4131.42625.725.72717.717.72828.028.02915.915.93016.516.5C=O171.0170.9CH321.321.3Epifriedelanol is a triterpenoid natural product previously isolated from the root bark ofVitis trifolia with antitumor activity [14] and also obtained from Anoectochilus chapaensis showing protein tyrosine phosphatase 1B inhibiting property [15]. Ulmus davidiana is also a source of epifriedelanol potent to reduce cellular senescence in human primary cells that control tissue aging or aging-associated diseases [11]. Quercus variabilis [16] also contains this bioactive metabolite. Anticancer activity of the compound isalso reported [17]. According to the report [12], I. confertiflora is rich in sesquiterpene lactones such as graveolide, carabrone, and carpesiolin. In the course, the occurrence of stigmasterol and thymol was also verified. In relation to these metabolites, different studies [3, 4] reported the isolations and characterizations of graveolide from I. graveolens, I. hupehensis, I. sericophylla, I. hookeri, and I. falconeri; carabrone from I. viscosa, I. falconeri, I. cappa, I. hookeri, I. royleana, I. hupehensis, and I. helenium; and carpesiolin from I. hupehensis, I. falconeri, I. sericophylla, and I. hookeri. Similar phytochemical investigations also show the presence of stigmasterol in I. helenium, I. cappa, and I. salsoloides [3, 4, 18] and thymol and its derivatives in I. helenium, I. cuspidate, I. cappa, and I. ensifolia [3, 4]. To the best of the author’s knowledge, epifriedelanol and dammara-20,24-dien-3-yl acetate are not ubiquitous in all species of the genus and they are previously reported only from I. cappa [3, 4]. In contrast, Ethiopian I. confertiflora contained these rare bioactive natural products.


## 6. Conclusion

Chemical investigations on ethanol crude extract ofI. confertiflora root afforded two rare triterpenes namely, epifriedelanol and dammara-20,24-dien-3-yl acetate. In the same study, the presence of stigmasterol and thymol was also noticed in the plant’s root. This study confirms the potential of I. confertiflora as the source of bioactive compounds of human importance related to other members of the genus.



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*Source: 2114595-2022-08-30.xml*