Bioactive non-sterol triterpenoid from Streblus asper: microwave-assisted extraction, HPTLC profiling, computational studies and neuro-pharmacological evaluation in BALB/c mice

Figures & data

Figure 1. Chemical structures of some non-sterol triterpenes from the plant S. asper.

Table 1. Preliminary phytoprofile and percentage yield of crude extract and various fractions of the stem bark of S. asper Lour.

Extract/fraction	Colour	Physical state	Weight (g)	Percentage yield (w/w)
Crude extract	Dark brown	Sticky solid	5.96	3.96
n-Hexane fraction	Dark green	Sticky solid	1.86	1.24
Dichloromethane fraction	Brown	Powder	1.02	0.68
Aqueous fraction	Reddish brown	Powder	2.69	1.79

Table 2. Results of phytochemical tests of crude extract and various fractions of the stem bark of S. asper.

Class of compounds	Crude extract	n-Hexane fraction	Dichloromethane fraction	Aqueous fraction
Alkaloids	+	+	+	−
Steroids	+	+	+	−
Flavonoids	−	−	−	−
Saponins	−	−	−	−
Glycosides	+	+	+	+
Amino Acids	+	−	+	+
Proteins	+	−	+	+
Phenolic Compounds	+	+	−	−
Fixed Oils	+	+	−	−
Carbohydrates	+	−	−	+

Indication: (+) = Present; (−) = Absent.

Figure 2. Spectral characterization of the isolated compound: (a) UV spectrum; (b) IR spectrum; (c) mass spectrum; (d) ¹H NMR spectrum and (e) ¹³C NMR spectrum.

Table 3. Observed characterization details and predicted computational data of the isolated non-sterol triterpenoid, betulin.

Observed characterization details		Predicted computational details
Characterization parameters	Remarks	Parameters	Values (reference values)
Physical state	Solid	Glide XPgscore	−2.09 (4COF Co-crystal ligand: 4.89)
Colour	White	Molecular weight	442.724 (130 to 725)
Phytochemical test	Positive results for Libermann Burchard and Salkowski tests.	H-bond donor (donorHB)	2 (0 to 6)
Per cent yield	0.046% w/w	H-bond acceptor (accptHB)	3.4 (2 to 20)
Melting range	252–254 °C	Oil/water partition coefficient (QlogPo/w)	5.87 (−2 to 6.5)
Solubility	Methanol, chloroform, n-hexane	Brain/blood partition coefficient (QPlogBB)	−0.369 (−3 to 1.6)
Rf value	0.33 (corresponds to the Rf value of the standard betulin, 0.32	Predicted central nervous system activity (CNS)	0 (−2 to +2)
IR (ν, cm^−1, KBr method) spectrum	1643.72 cm^−1 (C = C), 3095.04 cm^−1 (C–H), 1222.94 cm^−1(C–O), 3627.47 cm^−1(O–H)	Number of likely metabolic reactions (metab)	4 (1 to 8)
^1H NMR (300 MHz, CDCl3, δ, ppm) spectrum	1.076–1.131 (s,18H, CH3), 1.256–1.380 (s, 22H, CH2),1.605–1.638 (s, 8H, CH), 4.509–4.569 (m, 2H, OH)	% Human Oral absorption	100 (>80% is high; <20% is poor)
^13C NMR (300 MHz, CDCl3, δ, ppm) spectrum	21.16 (–CH3), 30.05 (–CH2), 39.86(=CH–), 55.48 (C–OH)	Prediction of binding to human serum albumin (QPlogKhsa)	1.497 (−1.5 to 1.5)
Mass spectrum	(M + 1) peak at m/z: 443.24 (C30H50O2: 442.72). Molecular ion peak at m/z 425.24, corresponded to protonated betulin dehydration product [M − H2O]^+	Volume (total solvent accessible volume in cubic angstrom using a probe with 1.4 Å radius)	13.86.34 (500 to 2000)
Elemental analysis	Calc.: C – 81.390; H – 11.380; Found: C – 81.894; H – 11.729%	Polar surface area (van der Waals polar surface area of nitrogen and oxygen atoms)	40.902 (7 to 200)
–	–	Number of violations of Lipinski’s rule of five	1 (Maximum 4)

Figure 3. (a) Calibration curve of standard betulin working standard solution; (b) 3D spectra of betulin working standard solutions (tracks 1–5), crude extract (track 6), n-hexane fraction (track 7) and isolated compound (track 8) scanned at 295 nm wavelength.

Figure 4. Fingerprints of (a) crude extract; (b) n-hexane fraction and (c) isolated compound.

Figure 5. HPTLC chromatogram of (a) crude extract; (b) n-hexane fraction and (c) isolated compound, against standard biomarker, betulin.

Table 4. Data showing quantity of betulin in crude extract, n-hexane fraction and isolated compound of S. asper.

Extract or fraction	Amount of betulin (mg/g)	Amount of betulin (%)
Crude extract	157.14	15.71
n-Hexane fraction	265.27	26.53
Isolated compound	922.41	92.24

Table 5. Data showing R_f range and area under the curve of standard betulin (Tracks 1–5), crude extract (Track 6), n-hexane fraction (Track 7) and isolated compound (Track 8).

S. No.	Concentration (μg/mL)	Start position	Maximum Rf	End position	AUC
Track 1	20	0.80	0.89	0.92	5257.5
Track 2	40	0.80	0.90	0.92	5311.8
Track 3	60	0.80	0.90	0.93	5394.1
Track 4	80	0.80	0.90	0.93	5472.8
Track 5	100	0. 80	0.91	0.93	5583.0
Track 6	1000	0.80	0.90	0.93	5223.8
Track 7	1000	0.80	0.90	0.93	5267.7
Track 8	1000	0.80	0.90	0.92	5534.5

Table 6. Data showing anticonvulsant, antidepressant and neurotoxicity studies of the crude extract, various fractions and isolated compound from S. asper Lour.

			Anticonvulsant activity									Antidepressant activity
			MES induced seizure model						Isoniazid induced seizure model			FST		TST		Neurotoxicity study
			Duration of various phases (s)				Recovery or death	Per cent inhibition of seizure	Duration of phases (s)	Recovery or Death	Per cent inhibition of seizure	Duration of immobility (s)	Per cent reduction in immobility	Duration of immobility (s)	Per cent reduction in immobility	Mean fall-off time after drug treatment (s)
Groups	Treatment	Dose (mg/kg b.w.)	Flexor	Extensor	Clonus	Stupor
1.	Control (CMC)	0.5%	3.69 ± 0.50	14.80 ± 0.86	40.85 ± 1.46	253.43 ± 1.57	Death	–	57.41 ± 0.93	Death	–	183.40 ± 1.44	–	205.60 ± 1.32	–	276.21 ± 5.29
2.	Standard (Diazepam/Floxetine)	520	2.26 ± 0.49	0.64 ± 0.25	6.44 ± 0.93	5.81 ± 1.02	Recovery	95.95	3.20 ± 0.37	Recovery	94.42	62.42 ± 0.93	65.79	92.82 ± 0.93	54.96	192.20 ± 6.19
3.	Crude Extract	100	5.21 ± 0.58	9.41 ± 0.40	–	–	Death	36.49	43.41 ± 1.18	Death	24.39	145.22 ± 1.30	20.82	190.81 ± 1.24	10.12	281.44 ± 4.66
		200	3.42 ± 0.51	6.48 ± 0.51	66.2 ± 2.59	168.41 ± 1.81	Recovery	56.76	27.20 ± 0.34	Recovery	52.61	130.61 ± 1.21	28.79	183.70 ± 0.83	10.65	287.62 ± 4.63
		400	2.20 ± 0.37	4.83 ± 0.49	38.22 ± 1.43	139.65 ± 1.63	Recovery	67.57	20.00 ± 0.32	Recovery	64.81	128.55 ± 0.48	29.91	160.28 ± 0.37	22.08	309.40 ± 7.53
4.	n-Hexane Fraction	100	4.48 ± 0.93	3.88 ± 0.37	27.40 ± 2.38	181.23 ± 2.22	Recovery	74.32	21.86 ± 0.49	Recovery	62.02	105.43 ± 0.76	42.51	156.24 ± 1.43	24.03	267.82 ± 3.28
		200	4.40 ± 0.93	2.63 ± 0.51	31.85 ± 1.53	179.85 ± 5.03	Recovery	82.43	15.23 ± 0.58	Recovery	73.52	89.40 ± 1.21	51.25	154.12 ± 0.84	25.04	301.45 ± 5.84
		400	3.47 ± 0.75	1.80 ± 0.37	27.41 ± 2.82	176.40 ± 3.71	Recovery	87.84	6.45 ± 0.51	Recovery	88.85	72.60 ± 0.34	63.50	122.15 ± 1.11	40.42	298.23 ± 7.86
5.	Dichloro-methane Fraction	100	5.08 ± 0.45	5.4 ± 0.75	42.89 ± 1.77	150.44 ± 1.66	Recovery	63.51	28.08 ± 0.45	Death	51.22	97.24 ± 1.49	46.98	143.57 ± 1.70	30.17	302.80 ± 4.75
		200	4.68 ± 0.46	4.26 ± 0.24	28.60 ± 1.44	140.80 ± 1.66	Recovery	71.62	19.61 ± 0.68	Recovery	65.85	99.05 ± 1.00	46.02	128.0 ± 1.32	37.74	295.87 ± 5.25
		400	4.23 ± 0.74	2.85 ± 0.37	25.80 ± 1.46	131.47 ± 2.02	Recovery	81.08	10.43 ± 0.40	Recovery	81.88	82.44 ± 0.88	55.05	96.12 ± 0.87	53.25	294.46 ± 4.80
6.	Aqueous Fraction	100	5.68 ± 0.81	10.84 ± 0.37	–	–	Death	27.03	47.66 ± 0.25	Death	17.07	178.36 ± 1.25	2.75	187.45 ± 1.21	7.19	277.23 ± 4.84
		200	4.85 ± 0.37	9.69 ± 0.68	–	–	Death	35.14	32.21 ± 0.66	Death	43.91	152.8 ± 1.77	16.69	185.33 ± 0.21	8.83	290.62 ± 5.10
		400	3.62 ± 0.40	7.02 ± 0.55	43.85 ± 1.86	221.81 ± 2.71	Recovery	52.70	24.46 ± 0.81	Recovery	57.49	130.24 ± 0.67	28.99	184.80 ± 1.72	9.86	315.28 ± 7.47
7.	Isolated compound	25	3.89 ± 0.75	8.25 ± 0.37	–	–	Death	44.60	32.27 ± 0.66	Death	43.90	100.23 ± 1.33	45.34	164.77 ± 0.93	19.86	321.46 ± 7.71
		50	5.27 ± 0.49	4.43 ± 0.25	6.81 ± 1.30	167.25 ± 1.63	Recovery	70.27	17.80 ± 0.58	Recovery	68.99	93.65 ± 0.71	48.94	156.61 ± 1.28	23.82	306.49 ± 6.26
		100	2.40 ± 0.51	2.22 ± 0.20	8.43 ± 1.77	–	Recovery	85.14	9.64 ± 0.40	Recovery	83.18	77.50 ± 1.21	57.54	132.53 ± 1.05	35.54	274.88 ± 3.85

The values in bold signify the most active fraction/compound.

Figure 6. Binding modes of the isolated compound betulin at the GABA_A receptor target protein: (a) 2D ligand interaction diagram; (b) 3D ligand interaction diagram, showing ligand–receptor H-bond interactions.