Phytochemical investigation of plant material is important because of its therapeutic actions. Several useful bioactive phytoconstituents were isolated from the plants sources such as digoxin, reserpine, taxol, and vincristine, quercetin etcthat has different pharmacological properties^1-2. Different species and different parts of a particular plant such as leaves, bark, root, stem, pod, seeds and fruit would have different constituents. Plants have been used as sources of a drug due to different biological activities³, Triterpenoids play a very important role in a plant’s defence mechanism⁴. They protect the plants from both constitutive and induced defensive responses against insects and environmental stress. Alternatively, the literature also describes some relationships between pentacyclic triterpenoids and treatments for Alzheimer's disease AD ^5-7

Boehmeria rugulosa, which is commonly known as “Gathi” in Hindi, is a medium-sized tree and distributed in the Himalayan region. This plant traditionally used for various purpose such as bone fracture, diabetes militias and also a source of fodder, wood also used for making various vessels. Therefore, the present work describes a phytochemical constituent’s study of the bark of B. rugulosa that was directed to the isolation of triterpenes.

The phytoconstituents isolated from the plant material are secondary metabolites and their derivatives like flavonoids, saponins, quinines, terpenoids, steroids, alkaloids, anthroquinones, and phenolic compounds etc, which are used as crude drugs^8-9 for medicinal purpose, several biologically active alkaloids, phenolic compounds have been isolated from some species of Boehmeria (Urticaceae)^10-15. Species of the genus Boehmeria are used in the traditional medicine for the treatment of Anti-hepatitis B¹⁶ and anti-inflammatory and antitumor activity¹⁷, antimicrobial and anti-diabetic activity¹⁸ this Plants can significantly contribute in human food because plants contain essential nutrients¹⁹ and for other purposes.

In the present work we have investigated the from ethyl acetate extract of bark of B. rugulosa and isolated triterpenoids namely (Fig. 1).

Fig. 1 (a.) R₁ = O, R₂ = H, R₃ = COOH, R₄ = CH₃

(b.) R₁ = O, R₂ = H, R₃ = CH₃, R₄= COOH

(a.)3-oxo-20-demethylisoaleuritolic-28,29-dioic acid.

(b.) 3-oxo-20-demethylisoaleuritolic -28,30-dioic acid.

MATERIAL AND METHOD:

Reagents and Chemicals:

Ultra-violet absorption spectrum was recorded on (Shimadzu UV-18000). Infrared spectrum was recorded on (Perkin Elmer Spectrum one) by Attenuated total reflection (ATR). ¹H and ¹³C were recorded on Avance-II-400MHz Bruker (Ultrashield^TM) instrument using deuterated chloroform (CDCl₃) as a solvent and TMS used as an internal standard for an examined compound. Mass spectra were recorded on LC-MS/MS (waters) and HPTLC (Camag). Petroleum ether (40-60^oC) (Fisher Scientific), chloroform (Fisher Scientifics), ethyl acetate (Fisher Scientifics), silica gel for column chromatography of mesh 100-200 (Merk), silica gel-G for TLC (Merk), silica gel-60 and silica gel F₂₅₄(0.25 mm layer thickness pre-coated on aluminium back) (Merk), iodine resublimed (CDH) and silica gel-G F₂₅₄ (Rankem) were purchased. All the other solvents and chemical used were of HPLC and analytical grade.

Plant material:

The bark of B. rugulosa was collected from the Rishikesh region. Voucher specimens have been put in the Herbarium of the Botanical Survey of India, Dehradun, in November 2015, with accession no. 115901.

Extraction and isolation:

The air-dried and powdered plant material (bark) was submitted to extraction with successive solvents (petroleum ether, ethyl acetate, acetone and ethanol) by soxhlet extraction method. The crude ethyl acetate extract (15g) was subjected for column chromatography packed with silica gel (100-200 mesh) and elution was carried with a gradient of petroleum ether-ethyl acetate from the ratio (100:0 to 90:1, 95:5, 90:10 85:15, 80:20 v/v). Similar fraction was pooled together on the basis of TLC behaviour. One compound was isolated from fraction number (15-25), while eluting the column with petroleum-ether: ethyl acetate (90-10) as white amorphous powder and another was isolated from fraction (30-45) with gradient petroleum ether: ethyl acetate. These compounds were isolated for the first time from B. rugulosa bark.

LC-MS QTOF analysis of ethyl acetate extract of bark

Analysis was performed on a Xevo G2-XS Qtof of waters with acquity UPLC- I Class and Unifi software. Separation was carried out using Acquity UPLC HSS-T3 column (100×2.1mm, 1.7µm). The column was maintained at 40^oC throughout the analysis and sample temperature was kept at 20^oC during the analysis. Detection was carried out by Xevo G2-XS Qtof. The elution was carried out at a flow rate of 0.4ml/min using gradient elution of mobile phase 0% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). 1µl of test solution was injected during the analysis and recorded the chromatogram for 45 min.

Fig. 2 LC-MSQt of chromatogram of ethyl acetate fraction of bark of B. rugulosa (a.) 3-oxo-20-demethylisoaleuritolic-28,29-dioic acid. (b.) 3-oxo-20-demethylisoaleuritolic -28,30-dioic acid.

Compound 1 was obtained as a white amorphous powder. The high resolution mass spectrum established the molecular weight with the molecular ion at m/z 485.2931 (requires 485.2931) and the molecular formula as C₃₀H₄₆O₅. The ¹H NMR data indicated that the compound was a terpenoid with six tertiary methyl groups at δ 0.80-1.03 (s, 3H each). The ¹³C NMR spectrum exhibited the presence of two carboxyl groups δ178.7 assigned to the C-17 carboxyl, and the other carboxyl signal at δ 181.0 might be assigned to either C-29 or C-30 due to the fragment of ion in the ESl-mass spectrum. Comparing with the related compounds²⁰, the carboxylic group (δ 181.0) should be located at the C-29 position. From these results and compared with the related data in reference the compound was identified as 3-oxo-20-demethylisoaleuritolic-28,29-dioic acid.

Compound 2 is an amorphous powder. The ESI-mass spectrum indicated the molecular weight was 485.34 (requires 485.34) and the molecular formula was established as C₃₀H₄₄O₅. The ¹H NMR spectrum suggested that the compound was a triterpenoid with six tertiary methyl groups and two carboxylic acids (12.15, 2H, Hz). ¹³C NMR spectrum exhibited the presence of two carboxylic carbons (176.8, 178.7). Comparing its ¹³C NMR data with those of compound 1, there was 3-ketonic carbon (δ 119.2) as in 1. The characteristic peaks at m/z 273, 265, 207 and 177 in the ESl-mass spectrum indicated the a methyl group at C-8, a ketonic group at C-3 and a carboxylic group at either C-29 or C-30 (15-16). In the mass spectrum of compound, the m/z 289 fragments confirming the presence of C-8 methyl group. The carboxylic carbon signal at δ 178.6 was typical for a C-17 carboxyl group therefore the other carboxylic group (signal at δ 176.8) was then assigned to C-30 due to this carboxyl group being axial orientated (19-20). Based on these results, the structure of the new compound was determined to be 3-oxo-20-demethylisoaleuritolic-28, 30-dioic acid.

Compound 1. Amorphous powder, soluble in CHC1₃. TLC (silica gel): Rf 0.58 (pet ether: EtOAc). Found: MW 486.3185, C₃₀H₄₆0₅, IR v_max2925 (C-H), 2854 (C-H), 1710 (C=O), 1464(gem –CH₃), 1382 (-CH₃), 1183, 1083, 904 and 725 (cm^-1). UV λ_max 258 nm. ¹H NMR (CDCl₃, 400 MHz): 0.84, 0.89, 0.90, 1.00, 1.01, 1.05, (3H, each, s, tert-Me). ¹³C NMR (CDCl₃, 400 MHz): δ16.27, 18.19, 21.45, 23.30, 26.96, 33.15 (δC, tert-Me); 178.7 (C-17-COOH); tert-methyl 181.0 (C-20-COOH); 119.2 (C-3); CH₂: 19.04, 23.30, 24.17, 24.69, 32.38, 33.94, 34.36, 36.08, 39.49, 43.56; CH: 43.63, 46.41, 55.04, quaternary carbon: 30.95, 37.00, 39.51, 47.61, 56.03. ESI/MS m/z: 485, 459, 425, 351, 337, 273, 265, 205, 189, 177, 149.

Compound 2. Amorphous powder, soluble mixture of CHCI₃, TLC (silica gel): Rf 0.55 (pet ether: EtOAc, 9:1). Found MW 486.3342, C₃₀H₄₆O₅, IR v_max2925 (C-H), 2854 (C-H), 1710 (C=O), 1464(gem –CH₃), 1382 (-CH₃), 1183, 1083, 904 and 725 (cm^-1). UV λ_max 258 nm. (pet ether: EtOAc, 8:15). ¹H NMR (CDCl₃, 400 MHz): tert Me (s): 0.75, 0.80, 0.81, 0.82, 0.86, 0.87, (1H), 4.18 (1H, H-3/3), 3.15 (1H, H-13), 12.15 (2H, COOH-28, 30). ¹³C NMR (CDCl₃, 400 MHz): 33.11 (C-1), 23.98 (C-2), 220.06 (C-3), 37.11 (C-4), 46.40 (C-5), 25.45 (C-6), 33.54 (C-7), 36.90 (C-8), 48.40 (C-9), 36.90 (C-10), 18.00 (C-11), 40.18 (C-12), 46.42 (C-13), 23.90 (C-16), 55.35 (C-17), 43.01 (C-18), 31.85 (C-19), 30.57 (C-20), 36.17 (C-21), 22.58 (C-22), 33.25 (C-23), 18.20 (C-24), 16.10 (C-25), 22.50 (C-26), 23.60 (C-27), 178.6 (C-28), 28.80 (C-29), 176.8 (C-30). ESI-MS m/z 485, 486, 459, 427, 273, 189.

HPTLC analysis of isolated compounds (3-oxo-20-demethylisoaleuritolic-28,29-dioic acid and 3-oxo-20-demethylisoaleuritolic -28,30-dioic acid):

The R_f 0.58 (Peteroleum ether-ethyl acetate 95:15, 90:10 v/v). High-performance Thin Layer chromatography separation of target analytic compound 1 was performed on 10cm × 10cm aluminum-backed HPTLC plates coated with 200µm layers of silica gel 60F₂₅₄ (E. Merck, Darmstadt, Germany). Before using the plates were prewashed with methanol and activated at 110°C for 5 min. Samples (5µL each) were applied on to HPTLC plate as 6mm wide bands and 12mm apart from the middle of bands by spray-on technique along with nitrogen gas supply for simultaneous drying of bands, by means of a Camag (Switzerland) [20]. the fingerprinting of compound I was developed on silica gel using mobile phase petroleum ether, formic acid, acetic acid, ethyl acetate 90: 11:11:10 (v/v/v). One triterpene was observed under 254nm and 366nm with R_f0.58 observed in fig 3 and after derivatization with anisaldehyde reagent was carried out and developed TLC shown in fig 4.

High-performance Thin Layer chromatography separation of target analyte compound 2 was performed on 10cm × 10cm aluminum-backed HPTLC plates coated with 200µm layers of silica gel 60F₂₅₄ (E. Merck, Darmstadt, Germany). Before using the plates were prewashed with methanol and activated at 110°C for 5 min. Samples (5 µL each) were applied on to HPTLC plate as 6mm wide bands and 12mm apart from the middle of bands by spray-on technique along with nitrogen gas supply for simultaneous drying of bands, by means of a Camag (Switzerland). the fingerprinting of compound I was developed on silica gel using mobile phase petroleum ether, formic acid, acetic acid, ethyl acetate 70: 11:11:10 (v/v/v). One triterpenoid was observed under 254nm and 366nm with Rf 0.61 observed in fig 5 and after derivatization with anisaldehyde reagent was carried out and developed TLC shown in fig 6.

Fig 3: HPTLC chromatogram of isolated compound 1 with Rf value 0.58.

Fig 4. HPTLC fingerprinting after derivatization for compound 1

Fig 5: HPTLC chromatogram of isolated compound 2 with Rf value 0.61.

Fig 6: HPTLC fingerprinting after derivatization for compound 2

CONCLUSIONS:

The ethyl acetate extract of the bark of B. rugulosa provided two triterpenoids. These two triterpenoids isolated first time from the bark of this plant. The LC-MSQtof chromatogram of fraction and resulting from selected compound and identification was carried out with the combination of experimental NMR (¹H & ¹³C), IR and LC-MSQtof analyses were a useful procedure for the structural determination of these triterpenoids²¹.

ACKNOWLEDGMENT:

We wish to acknowledge the Department of Kanya Gurukula Campus, Gurukula Kangri University, Haridwar, for providing all research facilities in the present study.

CONFLICTS OF INTEREST:

There are no conflicts of interest.

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Received on 07.04.2020 Modified on 16.11.2020

Research J. Pharm. and Tech. 2021; 14(6):2919-2923.

DOI: 10.52711/0974-360X.2021.00512