INTRODUCTION:

Tectona grandis is commonly known as the Teak tree. Being a tough and water-resistant in nature, the teak wood stood inimitable in the timber industry¹. Teak is famous not only for its uniqueness to produce long lasting furniture but also as a folklore medicine. Extensive literature survey about the phytochemistry and pharmacology of Tectona grandis reveals its antibacterial², antifungal³, antioxidant⁴, analgesic, anti-inflammatory⁵, antidiabetic⁶, diuretic⁷, anti pyretic⁸, wound healing⁹, anti ulcer¹⁰, anti cancer¹¹ and hair growth promoting activities¹². Phytochemicals like glycosides, flavonoids, saponins, proteins, amino acids, carbohydrates, and tannins are also reported to present in the methanolic seed extract¹³.

Various injuries and diseases are most often presented with pain and fever. Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed drugs for their management but significant gastrointestinal complications like perforation, bleeding, peptic ulcers, and obstructions have limited their uses in clinical settings¹⁴.

Various traditional systems like Ayurveda have mentioned numerous plants to treat common ailments like fever. Present inclinations in the plant research extended to the fistful literature about the plants comporting antipyretic properties. In the present investigation, the teak seeds are screened for fever reducing potential in yeast induced pyrexia model in adult Wistar rats.

MATERIALS AND METHODS:

Plant material:

Tectona grandis fruits were collected in the month of February 2017 in Hyderabad and were identified by Dr. N. Sivaraj, Senior Scientist (Eco Botany), National Bureau of Plant Genetic Resources, Rajendranagar, Hyderabad, seed specimens have been deposited.

Extraction:

Five Kgs of the fresh seeds were shade dried at temperature 25-30ºC for 7 days. The dried seeds were powdered in a grinder. The alcoholic extract of the seeds were prepared by subjecting to repetitive maceration for 2 weeks with methanol after defatting with n-hexane. The extract was evaporated to dryness with rotary evaporator and lyophilized to get powder. The percentage of yield was calculated as 6% and the final methanolic extract was used for the analgesic activity studies.

Phytochemical screening:

By using standard experimental procedures, the methanolic seed extract of Tectona grandis were screened for the presence of various phytochemicals¹⁵.

Isolation:

The crude methanolic extract was subjected to column chromatography using silica gel (100-200 mesh) with various polarities using a blend of n-hexane, ethyl acetate and methanol with continuous monitoring with TLC. Column chromatography was repeated for several times to obtain pure compounds. After thorough screening, few major compounds were identified and isolated in the pure form. Crystallization technique was performed where ever it is necessary. Structures of isolated compounds were established with detailed spectral analysis using Mass, NMR and IR.

Experimental animals:

Adult wistar rats (8-10 weeks) were used for the pharmacological screening from Sainath agencies, Musheerabad, Hyderabad. Animals were randomly segregated into groups prior to the initiation of the experiment; the rats were acclimatized in polypropylene cage for an episode of 10 days under controlled temperature (26 ± 2ºC), relative humidity (45-55%) and dark/light cycle for a period of 12 hrs. Rodent pellet diet was supplied and water ad libitum. The institutional animal ethics committee (IAEC) has approved (CPCSEA/IAES/JLS/006/01/17/005) the study protocol before commencement of the experiment.

Acute toxicity studies:

The oral acute toxicity studies were also performed for the extract according to the OECD guidelines (Organization for Economic Cooperation and Development) 423 on albino rats with little modifications. A limit test dose 2000mg/kg for the rats was considered and it is considered that a dose of 1000mg/kg body weight was considered as safe¹⁶.

Antipyretic activity by Brewer’s yeast induced pyrexia Method:

A suspension of Brewer’s yeast (15%) in saline (0.9%) was prepared. Six groups each containing six rats of either sex were taken. The group I served as control, group II served as Standard (Paracetamol 150mg/kg), group III, IV and V served as test and received Tectona grandis methanolic seed extract of 200mg/kg, 400 mg/kg, and 600mg/kg respectively. A digital thermometer was inserted 2cm deep into the rectum and the rectal temperatures were recorded. After measuring the basal rectal temperature, the animals were fevered by injection of brewer’s yeast suspension (10mg/kg) subcutaneously in the back below the nape of the neck. The sight of injection was massaged in order to spread the suspension beneath the skin. Immediately after yeast administration, food was withdrawn, and the animals were returned to their housing cages. After 19 hrs of yeast injection the rise in rectal temperature was recorded. The dose of the test compound and standard drug was given orally. The rectal temperature was recorded again after 1, 2, 3 and 4 hrs. The various extracts were dissolved in saline with the help of Gum Acacia (2% w/v)¹⁷.

RESULTS AND DISCUSSIONS:

Phytochemical screening:

A range of qualitative phytochemical tests were executed to confirm the presence of glycosides, flavonoids, saponins, proteins, amino acids, carbohydrates and tannins.

Isolation:

Bioassay guided fractionation of the active fractions eluted with chloroform-methanol mixtures from silica gel chromatography led to the isolation of three compounds lupeol, ursolic acid and gallic acid along with β- sitosterol and β- sitosterol glucoside. Incidentally the flavonoids (I, II and III) have been isolated for the first time from this species.

Spectral data:

Spectral data of Compound 1:

Compound 1 is obtained as a colourless crystalline compound, ¹HNMR (CDCl₃, 300MHz) δ 3.52 (1H, m, H‐3), 5.35 (1H, m, H‐6), 5.16 (1H, m, H‐23), 4.65 (1H, m, H‐22), 3.6 (1H, m, H‐3), 2.29(1H, m, H‐20), 1.8‐1.87 (5H, m) ppm. Other peaks are observed at δ 0.76‐0.89 (m, 9H), 0.91‐1.05 (m, 5H), 1.35‐1.42 (m, 4H), 0.5‐0.70 (m, 3H), 1.8‐1.87 (m, 5H), 1.05‐1.19 (m,3H), 1.33‐1.62 (m, 9H) ppm. ¹³C NMR (CDCl₃, 100MHz) has given signal at 140.77 (C‐5), 121.73(C‐6), 71.82 (C‐3), 56.80 (C‐17), 56.19 (C‐14), 50.16 (C‐9), 42.32 (C‐24), 42.35 (C‐13), 39.82 (C‐4), 39.55 (C‐12), 37.29 (C‐1), 36.53 (C‐10), 36.22 (C‐8), 35.82 (C‐20), 31.94 (C‐22), 31.68 (C‐7), 28.27 (C‐8), 28.04 (C‐25), 24.33 (C‐16), 23.87 (C‐2), 22.85 (C‐15), 22.60 (C‐28), 21.12 (C‐11), 19.43 (C‐26), 18.75 (C‐27), 11.90 (C‐19). IR: 3409.15 cm^‐1 (O‐H stretching.); 2932.62 cm^‐1 and 2866.44 cm^‐1 (aliphatic C‐H stretching); 1631.78 cm^‐1(C=C absorption peak); other absorption peaks includes 1465.98 cm^‐1 (CH2); 1376.53 cm^‐1 (OH def), 1058.77cm^‐1 (cycloalkane) and 957.01 cm^‐1 ; M⁺peak was observed at 414 corresponding to the molecular formula C₂₉H₅₀O which confirms that the compound is beta sitosterol (Figure 1).

Figure 1. structure of compound 1 (beta sitosterol)

Spectral data of Compound 2:

The compound was isolated as white crystalline compound with needle shapes. ¹HNMR (CDCl₃, 400MHz) δ 4.70 (2H, s, H-29a), 4.55(2H, s, H-29b), 3.2(1H, m, H-3), 2.37 (1H, m, H-19), 0.77 (3H, s), 0.79(3H, s), 0.85(3H, s), 0.94(3H, s), 0.97(3H, s), 1.15(3H, s), 1.85 (3H, s); ¹³C NMR (CDCl₃,100MHz): δ 150.94(C-20), 109.3(C-29), 78.9(C-3), 77.3(C-5), 77(C-9), 76.8(C-18), 55.3(C-19), 50.4(C-17), 48.3(C-14), 47.9(C-8), 42.9(C-22), 40(C-4), 38.5(C-1), 38.7(C-13), 38 (C-10), 37.1(C-16), 35.6(C-7), 34.3(C-21), 29.8(C-23), 27.9(C-2), 27.4(C-15), 27.4(C-12), 25.3(C-11), 20.9(C-30), 18.3(C-6), 18.0(C-28), 16.1(C-25), 15.9(C-26), 15.3(C-24), 14.5(C-27). IR v max (CCl4)cm-1: 3056, 2929, 2313, 1593, 1435, 1265, 898, 741; M⁺peak was observed at 426 corresponding to the molecular formula C₃₀H₅₀O.

From the ¹H NMR spectrum data, seven 3° methyl protons were identified as an integration of three each at δ 0.77, 0.79, 0.85, 0.94, 0.97, 1.05 and 1.65. A characteristic sextet peak was also observed at δ 2.37 which is represents 19β –protons. The H-3 proton showed a multiplet at δ 3.2 while a pair of broad singlets at δ 4.55 and δ 4.70 (IH, each) was indicative of olefinic protons at (H-29a & b) which confirms that the compound is lupeol.

From ¹³C NMR data, seven methyl groups were observed at δ: 29.8 (C-23), 18.0 (C-28), 16.1 (C-25),15.9 (C-26), 15.3 (C-24), 14.5 (C-27) and 20.9 (C-30). Peaks at δ: 109.3 (C-29) and 151.0 (C-20) represents exomethylene groups and also found ten methylene carbons, five methine and five 4° carbons. The deshielded signal at δ 79.0 was due to C-3 with a hydroxyl group attached to it.

IR spectrum showed characteristic peaks at 3056 and 1265cm^-1 typical of the O-H and C-O bond vibrations, respectively;peaks at 898cm^-1 was due to an unsaturated out of plane C-H vibration; the C=C vibrations was shown around 1593cm^-1 as weakly intense band; stretching and bending vibrations due to methyl groups were represented by the bands at 2929cm^-1 and 1593cm^-1 andthe signal at 1435cm^-1 was due to methylenic vibration. M⁺peak was observed at 426 corresponding to the molecular formula C₃₀H₅₀O which confirms that the compound is lupeol (Figure 2).

Figure 2: structure of compound 2 (lupeol)

Spectral data of Compound 3:

The compound was isolated as white crystalline compound.1H-NMR (500 MHz, CDCl₃): δ 5.47 (1H, brs, H-12), 3,44 (1H, dd, J = 10.0, 5.5 Hz, H-3), 2.62 (1H, d, J = 11.0 Hz, H-18), 2.31 (1H, td, J = 13.0, 3.5 Hz, H15), 2.10 (1H, td, J = 13.5, 4.0 Hz, H-16), 1.23 (3H, s, Me-23), 1.21 (3H, s, Me-27), 1.03 (3H, s, Me-26), 1.01 (3H, s, Me-24), 0.98 (3H, d, J = 6.0 Hz, Me-29), 0.95 (3H, d, J = 6.0 Hz, Me-30), 0.87 (3H, s, Me-25); ¹³C NMR (CDCl₃,100MHz): δ 38.90 (C1), 27.10(C2), 79(C3), 39(C4), 55.6(C5), 18.60(C6), 33.40(C7), 39.80(C8), 47.90(C9), 37.20(C10), 23.60(C11), 125.49(C12), 138.17(C13), 42.40(C14), 29.90(C15), 24.50(C16), 48.10(C17), 53.20(C18), 39.40(C19), 39.20(C20), 30.90(C21), 37.10(C22), 28.30(C23), 15.60(C24), 15.80(C25), 17.20(C26), 23.70(C27), 180.42(C28), 17.10(C29), 21.30(C30); IR v max (CCl4)cm-1: 2929.60, 1648.2, 1513.91, 1455.51, 1382.56,1313.29, 1186.26,1141.60 and 807.49. [M + H]⁺was observed at m/z 457 corresponding to the molecular formula C₃₀H₄₈O₃.

The carbon NMR showed carboxylic acid carbon at δ 180.42, two olefinic carbons at 125.49 and 138.17 and a saturated oxygen-bearing methane at 78.7.

Bonded –OH 2929.60 Bond C=O 1648.2 Aromatic 1513.91 C=C stretch aromatic 1455.51 -C-OH deformation vibrations 1382.56,1313.29 -C-OH stretching vibrations 1186.26,1141.60 9H-C– H out-of-plane bending 807.49; which confirms that the compound is ursolic acid (Figure 3).

Figure 3. structure of compound 3 (ursolic acid)

Spectral data of Compound 4:

The compound was isolated as white powder. 1H NMR δ 7.46 (s, 2H, ArH), 10.67 (s, 4H, -OH), 13C-NMR (DMSO-d6) ppm: δ 159.08 (C7), 148.08 (C4), 139.55 (C3), 136.35 (C2), 112.27 (C1), 110.21 (C5), 107.59 (C6); IR spectrum of the compound exhibit broad band in the range, 3476-3557 cm-1 which is attributed to the -OH stretching while the band observed at 1698 cm-1 corresponds to C=O stretching. The bands observed in the range, 1669 – 1500 cm-1 are due to aromatic ring vibrations. The band at 751 cm-1 is assigned to aromatic C-H bending vibration. The ESI-MS (negative mode) m/z is observed at 301.1 [M-H] corresponding to the molecular formula C₁₄H₅O₈ which confirms that the compound is ellagic acid (Figure 4).

Figure 4. structure of compound 4 (ellagic acid)

Spectral data of Compound 5:

It is obtained as a white amorphous powder. ¹H NMR [300 MHz, CD₃OD] δ = 7.06 (s, H-2 and H-6). δ ¹³C NMR [75 MHz, CD₃OD]: δ = 110.37 (C-2, C-6), 121.99 (C-1), 139.62 (C-4), 146.41 (C-3, C-5), 170.45 (COOH). The ESI-MS (negative mode) m/z is observed at 169 [M-H] corresponding to the molecular formula C₇H₆O₅which confirms that the compound is gallic acid (Figure 5).

Figure 5. structure of compound 5 (gallic acid)

Acute toxicity results:

Although traditional medicine is having advantages over the allopathic medicine, they also suffer with toxicity. Few naturally obtained compounds are also not safe to use without proper dosage regimen. Hence, toxicity evaluation is necessary for the safe use of plant based medicine. In this regard, the methanolic seed extract of T. grandis was evaluated for its toxicity in mice.

Results (Table 1) showed that, the extract is not showing any toxicity symptoms at a dose of 1000 mg/kg bodyweight orally and it is not safe to use the extract at higher doses.

Antipyretic activity by Brewer’s yeast induced pyrexia Method:

The antipyretic activity was assessed in male albino rats using paracetamol as a reference standard. The subcutaneous injection of yeast distinctly amplified the rectal temperature and the mean increment recorded was 1.24–2^∘F after 18 hr of administration.

The fall of temperature in test groups was statistically significant and it is in a dose dependent manner compared to the reference standard (Table 2 and Figure 6). Since the pattern of reducing temperature for the test sample was almost similar to that of standard, suggesting the antipyretic activity may be by interfering the prostaglandin production and other inflammatory mediators like cytokinines.

Table 1: Effect of T. grandis methanolic seed extract in albino mice

Group	No. of mice	Dose (mg/kg)	Body weight in gm			Outcome
Group	No. of mice	Dose (mg/kg)	Day 0	Day 7	Day 14	Outcome
1	5	Control	25	27	30	Survival (5/5)
2	5	10	26	28	30	Survival (5/5)
3	5	50	25	27	30	Survival (5/5)
4	5	100	24	25	29	Survival (5/5)
6	5	500	24	25	29	Survival (5/5)
7	5	1000	24	26	28	Survival (5/5)
8	5	1250	25	24	23	Survival (4/5)
9	5	1500	26	25	23	Dead (2/5)
10	5	2000	25	22	20	Dead (4/5)

Table 2: Antipyretic effect of methanolic seed extract of Tectona grandis on Brewer’s yeast induced pyrexia model.

Group	Rectal temperature in °C
	Before yeast administration	After treatment
	Before yeast administration	0hr	1hr	2hrs	3hrs	4hrs
Control	37.08±0.37	40.48±0.27	40.78±0.27	40.58±0.41	40.4±0.42	40.33±0.45
Paracetamol	37.25±0.42	40.3±0.2	39.58±0.17**	39.1±0.15**	38.41±0.36**	38.25±0.25**
Extract (200 mg/kg)	37.18±0.23	40.6±0.23	40.23±0.56	40.13±0.38*	39.85±0.30*	39.4±0.38**
Extract (400mg/kg)	37.2±0.54	40.48±0.24	40.1±0.28*	37.95±2.88*	37.35±2.88**	37.11±2.86**

Values are expressed in mean ± SEM. Data compared against positive control group. One way ANOVA, *P<0.05 **P<0.01 were considered statically significant when compared to control using Turkey-Kramer multiple comparison test.

Figure 6: Antipyretic effect of methanolic seed extract of Tectona grandis on Brewer’s yeast induced pyrexia model.

CONCLUSION:

To conclude, five compounds beta sitosterol, lupeol, ursolic acid, ellagic acid, and gallic acid were isolated from the methanolic seed extract of Tectona grandis and reported for the first time. The structures were established by detailed spectral analysis using sophisticated analytical techniques. From the pharmacological activity results, it can be affirmed that the seeds acquired considerable anti pyretic activity in adult Wistar rats in a dose dependent manner for the selected animal model. This study is discernible to have the antipyretic prospective of teak seeds and further investigation is obligatory to comprehend the molecular mechanism concerned in the antipyretic potential of Tectona grandis.

ACKNOWLEDGEMENT:

Authors are thankful to the department of Botany, Osmania University for providing necessary facilities.

CONFLICT OF INTEREST:

Authors declare that there is no conflict of interest.

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Received on 08.12.2019 Modified on 15.02.2020

Research J. Pharm. and Tech. 2021; 14(8):4221-4225.

DOI: 10.52711/0974-360X.2021.00732