INTRODUCTION:

Nigella sativa L. is an annual herbaceous plant of the Ranunculaceae family, 15 to 50 cm high¹. This is one of the most famous spicy-aromatic crops in the countries of the Mediterranean and Central Asia, which has a wide range of biologically active compounds and unique medicinal properties. In the Russian Federation, this and

another well-known species - Nigella damascene L., are often found in the southern regions of the European part of Russia, in the Caucasus and in the Crimea².

In the world scientific literature, there is a lot of information about the pharmacological activity of various groups of biologically active compounds contained in seeds and, accordingly, in Nigella oil: a high content of unsaturated fatty acids in fatty oil, a high content of thymoquinone and the presence of nigellon as components of essential oil, carbohydrates, and also lipolytic enzymes^1-9.

At present, Nigella sativa L. is not a pharmacopoeial plant, despite the clear interest of researchers in this species and the wide possibilities of its cultivation. Foreign literature reports on 15 compounds isolated and identified from the herb - mainly flavonoid (rutin, kaempferol 3-O-rutinoside, quercetin-3-gentiobioside, nigelflavonoside B, kaempferol-3,7-diglucoside, etc.) and triterpenoid nature (flacidoside III, α-hederin, 3-O-[α-1-rhamnopyranosyl-(1→2)-α-1-arabinopyranosyl] hederagenin, etc.), a new triterpenoid saponin glycoside has also been described¹⁰. However, in Russia, similar studies with cultivated Nigella sativa L. have not been previously conducted.

The antioxidant, antibacterial, and antidiabetic effects have been described for the herb, which may be due to the presence of the main group of biologically active compounds (BAC) - phenolic substances^11-18.

Therefore, it is necessary to conduct a complex of pharmacognostic studies, in particular, to conduct a phytochemical analysis in relation to substances of flavonoid nature in Nigella sativa L. herbs, taking into account its largest biomass among other raw plant organs.

The aim of this study was to study the phytochemical composition of Nigella sativa L. herbs and develop a method for quantitative determination of the sum flavonoid content in Nigella sativa L. herb by UV/VIS spectrophotometry.

MATERIAL AND METHODS:

Plant material:

The herb of the Nigella sativa L. was collected on the territory of the Samara region, in the Botanical Garden of Samara University during the period of mass flowering and the beginning of fruiting (early August 2020-2022).

Extraction of Plant Materials:

The extraction of the herb of Nigella sativa L. (190g) was carried out with the using of 70% EtOH in ratio 1:5, combining the maceration method with subsequent thermal extraction in a boiling water bath. The obtained water-alcohol extract was filtered and then evaporated by using a rotary evaporator at low temperature (40-50°C) and reducing pressure to thick residue (about 200 ml).

Isolation of Compounds from Plant Materials:

The isolation of compounds 1-5 from the obtained thick extract of the Nigella sativa L. herbs was carried out with the using of the method of adsorption column chromatography. For this the obtained concentrated extract was dried on L 50/100 silica gel and the resulting powder was applied to a silica gel layer formed in chloroform. The chromatographic column was eluted with chloroform and a mixture of chloroform-ethanol in various ratios (100:0, 97:3, 95:5, 93:7, 90:10, 85:15, 80:20, 70:30 and 60:40). The eluates were divided into fractions of approximately the same volume (200 ml each), then evaporated under vacuum. The elution of the compounds was monitored by TLC analysis on “Sorbfil PTLC-AF-A-UV” plates in a system of chloroform-ethanol-water (25:18:2). The spots of compounds on the chromatogram were detected by luminescence in UV light at a wavelength of 254 and 366 nm and by color visualization after processing the chromatograms with an alcoholic solution of aluminum chloride.

From the most promising fractions obtained by elution with a mixture of chloroform and ethyl alcohol at a ratio of 70:30 and 60:40, the dominant flavonoid substances 1-3 were isolated with an R_fvalue of about 0.48, 0.32 and 0.43, respectively. Besides, from the fractions where the eluate was a mixture of chloroform and ethyl alcohol in a ratio of 93:7, substances 4-5, presumably of a sterol nature, were also isolated.

Methods of Structural Elucidation of Flavonoids:

UV spectra were recorded using a spectrophotometer “Specord 40” (Analytik Jena, Germany) in cuvettes with a layer thickness of 10mm in the wavelength range from 190 nm to 600 nm. ¹H-NMR spectra were obtained on a spectrometer “JNM-ECX 400” (JEOL Ltd., Japan) at a frequency 399.78 MHz, ¹³C-NMR spectra were obtained on a spectrometer “JNM-ECX 400” (JEOL Ltd., Japan) at a frequency 100.52 MHz. Mass spectra were obtained on a Bruker micrOTOF II instrument by electrospray ionization (ESI).

UV/VIS Spectrophotometry of Plant Materials:

Differential spectrophotometry was used as a quantitative research method using an additional reagent -3% alcohol solution of AlCl₃. The UV spectra were recorded using a «Specord 40» spectrophotometer (Analytik Jena, Germany) in the wavelength range of 190–600 nm in cuvettes with a layer thickness of 10mm.

The raw material was crushed so that its particles passed through a sieve with holes 2 mm in diameter. An accurate weighed sample of ground material (about 1 g) was placed in a 100 mL flask and 30 mL of 70% ethyl alcohol was added. The flask was closed with a stopper and weighed on a balance accurate to 0.01 g. The flask was attached to a reflux condenser and heated in a boiling water bath for 45 min. After boiling, the flask was cooled for 30 min, closed with the same stopper, weighed again, and the extractant was added to its original weight. The resulting aqueous-alcoholic extract was filtered through a paper filter (solution of extract “A”). 1ml of the obtained extract “A” was placed in a 25 mL volumetric flask, 2 mL of a 3% alcoholic solution of aluminum chloride is added and the volume of the solution is adjusted to the mark with 96% ethyl alcohol (test solution). The optical density of the test solution was measured on a spectrophotometer at a wavelength of 410 nm 40 min after preparation of its. The comparison solution was a solution containing 1mL of solution of extract “A” and 96% ethyl alcohol, whichwas adjusted to the mark in a 25 mL flask. At the same time, the optical density of solution “B” of rutin was measured under the same conditions. As a comparison solution we used 2 ml of solution “A” of rutin, adjusted with 96% alcohol in a volumetric flask with a capacity of 25 mL.

The content of the total flavonoids in percent (X) calculated onrutin and absolutely dry raw materials of Nigella sativa L. was calculated by the formula:

Where: A is the optical density of the test solution;

А_o is the optical density of the solution “B” of rutin;

m is the exact weight of plant raw material, g;

m_o is the exact weight of rutin reference sample, g;

W is mass loss on drying, %.

Note:The preparation of rutin reference solution. About 0.02 g (accurately weighed of the reference sample of rutin (content of the main substance ≥98%) is placed in a volumetric flask with a capacity of 50 mL, dissolved in a small amount of 70% ethanol, diluted to the mark with 70% ethanol, mixed (solution “A” of rutin). 2 ml of solution “A” of rutin was placed in a 25 mL volumetric flask, 2 mL of a 3% alcohol solution of aluminum chloride is added and the volume of the solution is adjusted to the mark with alcohol 96% (solution “B” of rutin).

RESULTS AND DISCUSSION:

In the course of our research were isolated from the herb of Nigella sativa L. nicotiflorin (1), nigelflavonoside G (2), rutin (3), β-sitosterol (4), daucosterol (5) (Fig. 1), identified with using of UV, ¹H-NMR,¹³C-NMR spectroscopy and chemical transformations.

1 2

4 5

Figure 1: The chemical structures of compounds isolated from Nigella sativa L. herbs: nicotiflorin (1), nigelflavonoside G (2), rutin (3), β-sitosterol (4), daucosterol (5).

Nicotiflorin (kaempferol 3-O-rutinoside) (1). The crystalline substance is a light-yellow color composition С₂₇Н₃₀О₁₅; m.p. 182–186°C (water alcohol). l_mаx^EtOH 269, 355 nm; +NaOAc 272, 304, 365 nm; +АlCl₃ 274, 304, 345, 394 nm; + АlCl₃+HCl 275, 304, 345, 394 nm; + NaOMe 275, 326, 400 nm.

¹H-NMR spectrum (399.78 MHz, DMSO-d₆, δ, ppm, J/Hz): 12.52 (1H, br. s, 5-ОН-group), 10.10 (1Н, br. s, 7-ОН-group), 9.20 (1Н, br. s, 4’-ОН-group), 7.95 (2Н, д, J = 9.0, Н-2’ andJ = Н-6’), 6.83 (2Н, d, J = 9.0, Н-3’ and Н-5’), 6.36 (1Н, d, J = 2.5., Н-8), 6.15 (1Н, d, J = 2.5, Н-6), 5,25 (1Н, d, J = 7,0, Н-1’’of glucopyranose), 4,33 (1Н, d, 1.0 Hz, Н-1’”of rhamnose), 2.9-4.1 (10H, m,4H of rhamnose and 6H of glucose), 0,94 (3Н, d, 6.0 Hz, CH₃of rhamnose).

¹³C-NMR spectrum (100.52 MHz, DMSO-d₆, δ_C, ppm): 178.76 (С-4), 167.69 (С-7), 160.43 ( С-5), 157.04 (С-9), 148.44 (С-2 and С-4’), 133.72 (C-3), 131.39 (С-2’ and С-6’), 127.49 (С-1’), 115.18 (С-3’ and С-5’), 106.31 (С-10), 101.30 (С-1’’ of glucose), 99.97 (С-1’’’ of rhamnose), 99.35 (С-6), 94.33 (С-8), 76.90 (С-5’’ ), 76.24 (С-3’’), 74,07 (С-4’’’), 73.77 (С-2’’), 72.33 (С-4’’), 71.11 (С-5’’), 70.93 (С-5’’), 70.86 (C-2’’’), 70.43 (С-3’’ и C-3’’’ ), 70.10 (С-4’’), 68,92 (С-5’’’), 68,77 С-6’’), 18.25 (СН₃of rhamnose).

Mass spectrum (HR-ESI-MS, 180^оС, m/z): [M+H]⁺595.1164 m/z, [M+Na]⁺ 617.1003 m/z, [M+К]⁺ 633.0963 m/z.

Nigelflavonoside G (3-O-β-D-[α-L-O-rhamnopyranosyl (1→6)]-glucopyranoside-[(2→1)-O-β-D-glucopyranosyl (2→1)-O-β-D-glucopyranosyl]-3,5,6,7,4'-pentahydroxy-3'-methoxyflavone) (2). The amorphous substance is a yellow color composition С₄₀Н₅₂О₂₇. l_mаx^EtOH 272, 343 nm; + NaOAc 276, 340, 400sh nm; + NaOAc + H₃BO₃ 274, 340, 400sh nm; + АlCl₃ 282, 343, 415 nm; + АlCl₃ + HCl 282, 343, 410 nm; + NaOMe 280, 407 nm.

¹H-NMR spectrum(399.78 MHz, DMSO-d₆, δ, ppm, J/Hz): 12.60 (1H, br. s, 5-ОН-group), 9.0-10.4 (3Н, br. s, 6-ОН-group, 7-ОН- group and 4’-ОН-group), 7.52 (1Н, d, J = 2.5, Н-2’), 7.50 (1Н, dd, J = 2.5 and J = 8.5, Н-6’), 6.94 (1Н, s, Н-8), 6.85 (1Н, д, J = 8.5, Н-5’), 5.18 (1Н, d, 7.0 Hz, Н-1’’ of glucose), 4,64 (1Н, d, br. s, J = 7,0, Н-1’’Н-1’” of rhamnose), 4,55 (1Н, d, 7.0 Hz, Н-1’’’’ of glucose), 4,25 (1Н, d, 7.0 Hz, Н-1’’’’’ of glucose), 2.9-4.2 (22H, m, 18H of glucose + 4H of rhamnose), 3.72 (3H, s, 3’-ОСН₃), 0,83 (3Н, d, 6.0 Hz, 3H, CH₃ of rhamnose).

¹³C-NMR spectrum(100.52 MHz, DMSO-d₆, δ_C, ppm): 177.00 (С-4), 167.32 (С-7), 161.21 (С-5), 156.93 (С-2 and С-9), 148.46 (С-4’), 145.27 (С-3’), 135.00 (С-3), 131.00 (С-6), 121.00 (С-1’), 116.68 (С-2’ and С-6’), 115.76 (С-5’), 104.00 (С-10), 102.00 (С-1’’ of glucose), 101.06 (С-1’’’ of rhamnose, С-1’’’’ of glucose and С-1’’’’’ of glucose), 94.00 (С-8), 77.37 (С-3’’and С-5’’), 73.73 (С-4’’’, 72.85 (С-2’’ and С-4’’), 71.19 ( С-2’’’ and С-3’’’), 68.19 (С-5’’’ and С-6’’), 61.01 (С-6’’’’ and С-6’’’’’), 56.52 (3’–OCH₃), 18.28 С-6’’’ (СН₃of rhamnose).

Mass spectrum (HR-ESI-MS, 180^оС, m/z): [M+H]⁺965.2564 m/z, [M+Na]⁺ 987.2377 m/z, [M+К]⁺ 1003.2116 m/z.

Rutin (quercetin 3-O-rutinoside) (3). The crystalline substance is a yellow color composition С₂₇Н₃₀О₁₆; m.p. 192–194°C (water alcohol). l_mаx^EtOH 258, 266sh, 362 nm; + NaOAc 270, 303, 365 nm; + NaOAc + H₃BO₃ 270, 303, 380 nm; + АlCl₃ 273, 304, 360, 412 nm; + АlCl₃ + HCl 273, 304, 360, 400 nm.

¹H-NMR spectrum (399.78 MHz, DMSO-d₆, δ, ppm, J/Hz): 12.55(1H, s, 5-ОН-group), 10.63(1Н, br. s, 7-ОН- group), 9.60 (1Н, s, 4’-ОН-group), 9.20 (1Н, s, 3’-ОН-group), 7.51 (1Н, dd,2,5 and 9 Hz, Н-6’), 7.48 (1Н, d, 9 Hz, Н-2’), 6.80 (d, 9.0Hz, Н-5’), 6.34 (d, 2.5Hz, Н-8), 6.15 (d, 2.5Hz, Н-6), 5,30 (1Н, d, 7,0Hz, Н-1’’of glucose), 4,34 (1Н, d,br. s, Н-1’”of rhamnose), 2.9-3.8 (10H, m, 6H of glucose and 4H of rhamnose), 0,95 (3Н, d, 6.0 Hz, 3H, CH₃of rhamnose).

¹³C-NMR spectrum (100.52 MHz, DMSO-d₆, δ_C, ppm): 177.36 (С-4), 166.64 (С-7), 161.21 (С-5), 156.93 (С-2 and С-9), 148.94 (С-4’), 145.27 (С-3’), 135.74 (С-3), 122.10 (С-6’), 121.64 (С-1’ and С-2’), 115.64 (С-5’), 110.24 (С-10), 104.45 ( С-1’’ of glucose), 101,30 (С-1’’’ of rhamnose), 98.00 (С-6), 94.00 (С-8), 76.41 (С-5’’), 75.43 (С-3’’), 74.57 (С-4’’’, 72.34 (С-2’’ and С-4’’), 70.53 (С-2’’’ and С-3’’’), 68,76 (С-5’’’ and С-6’’), 18,25 С-6’’’ (СН₃of rhamnose).

Mass spectrum (HR-ESI-MS, 180^оС, m/z): [M+H]⁺611.1598 m/z, [M+Na]⁺ 633.1426 m/z, [M+К]⁺ 649.1165 m/z.

β-Sitosterol (4). The crystalline substance is a white color composition С₂₉Н₅₀О; m.p. 134-136°C (chloroform/hexane mixture).

¹H-NMR spectrum (399.78 MHz, DMSO-d₆, δ, ppm, J/Hz): 5,34 (1Н, m, H-6), 3,52 (1Н, m, H-3), 1.14-3.50 (30Н, m), 1.01 (6Н, s, CH₃-19 and CH₃-21), 0,83 (6Н, s, CH₃-26 and CH₃-29), 0,79 (3Н, s, CH₃-27), 0,68 (3Н, s, CH₃-18).

¹³C-NMR spectrum (100.52 MHz, DMSO-d₆, δ_C, ppm): 140.83 (С-5), 121.80 (С-6), 71.89 (С-3), 56.84 (С-14), 56.02 (С-17), 50.23 (С-9), 45.91 (С-24), 42.29 (С-13), 40.58 (С-12), 39.76 (С-4), 3734 (С-1 and С-22), 36.59 (С-10), 36.23 (С-20), 31.97 (С-2), 29.00 (С-25), 28.32 (С-16), 25.49 (С-23), 24.45 (С-15), 23.11 (С-11), 19.90 (С-26), 19.48 (С-19), 19.06 (С-27), 12.33 (С-18), 12.13 (С-29).

Daucosterol (β-sitosterol 3-O-b-D-glucopyranoside) (5). The crystalline substance is a white color composition С₃₅Н₆₀О₆; m.p. 312-315 °C (chloroform/ hexane mixture).

Mass spectrum (HR-ESI-MS, 180^оС, m/z): [M+Na]⁺ 599.1160 m/z, [M+К]⁺ 615.0899 m/z.

A spectrophotometric study of water-alcohol extracts from the herb of Nigella sativa revealed absorption maxima typical for substances of the flavonoid group^9-10: with direct spectrophotometry - 266±2 nm and 340±2 nm; after adding 3% alcohol solution of AlCl₃ - 275±2 nm, 340±2 nm, 408±2 nm; in the differential version (with AlCl₃) - 275±2 nm and 410±2 nm (Fig. 2 and 3).

Figure 2: The UV spectra of water-alcohol extraction from Nigella sativa L. herb.

Designations: 1 – extraction; 2 – extraction with AlCl₃.

Figure 3: The UV differential spectrum of water-alcohol extraction from Nigella sativa L. herb (with AlCl₃).

As can be seen from the presented data (Fig.2), when using a reagent specific for flavonoids (3% alcohol solution of AlCl₃), a characteristic bathochromic shift was observed from λ_max = 340±2 nm to λ_max = 408±2 nm, which repeats the pattern of the spectra of the standard rutin sample (Fig. 4). The differential spectrum also clearly repeats the corresponding extraction spectrum (Fig. 5). This allows a reference sample of rutin to be used to quantify the total flavonoid content of the extract according to the method developed by us at a wavelength of 410±2 nm.

Figure 4: The UV spectra of water-alcohol extraction from Nigella sativa L. herb and reference sample of rutin after added AlCl₃.

Designations: 1 – extraction with AlCl₃; 2 – rutin with AlCl₃.

Figure 5: The UV differential spectrum of reference sample of rutin (with AlCl₃).

Using the developed methodology, we analyzed a number of samples of N. sativa L. herbs for 2020-2022 (Table 1) and it was determined that the content of the total flavonoids varies from 1.17±0.16% to 1.72±0.22% (calculated onrutin).

Table 1 – The content of the sum of flavonoids in water-alcohol extraction from Nigella sativa L. herb (calculated on rutin).

No.	Plant raw material sample	The content of the total flavonoids calculated on rutin, %
1.	The herb of the N. sativa L. (Samara, the Botanical Garden of Samara University (early August 2020).	1,25±0,21
2.	The herb of the N. sativa L. (Samara, the Botanical Garden of Samara University (early August 2021).	1,17±0,16
3.	The herb of the N. sativa L. (Samara, the Botanical Garden of Samara University (early August 2022).	1,72±0,22

The metrological characteristics of the proposed UV/VIS spectrophotometry procedure indicate that the error in determining the average result of the sum of flavonoids (calculated on rutin) content in the herb of the N. sativa L. with a confidence level of 95% is ±4.17% (Table 2).

Table 3 - Metrological characteristics of the method for the quantitative determination of rutin in the herb of the N. sativa L.

*Sample*	f	𝑋*̅, %*	S	*P, %*	*t (P,f)*	ΔX	𝜀*̅, %*
Nigella sativa L. herb	10	1,17	0,073	95	2,23	±0,16	±4,17

Designations: f – degrees of freedom; 𝑋̅ – average; S – standard deviation; P – confidential probability, t - Student's t-test, ΔX– half-width of the confidence interval of the mean result; 𝜀̅ – mean relative error.

CONCLUSION:

As a result of the research, from the herb of Nigella sativa L. were isolated nicotiflorin, nigelflavonoside G, rutin, β-sitosterol, and daucosterol, chemical structural elucidation of which were carried out by means of UV, ¹H-NMR, ¹³C-NMR spectroscopy and mass spectrometry. A method for quantitative determination of the total flavonoids calculated on rutin in the herb of Nigella sativa L. was developed by spectrophotometry at a wavelength of 410 nm. The content of total flavonoids (calculated on rutin) in Nigella sativa L. herbs varied from 1.17±0.16% to 1.72±0.22% over a three-year period (2020-2022 years). This study contributes to the standardization of raw materials of the aerial parts of the plant for the subsequent integrated use of Nigella sativa L.

CONFLICTS OF INTEREST:

The authors declare no conflicts of interest.

REFERENCES:

1. Salehi B. Quispe C. Imran M. Ul-Haq I. Živković J. Abu-Reidah IM. et al. Nigella Plants - Traditional Uses, Bioactive Phytoconstituents, Preclinical and Clinical Studies. Frontiers in Pharmacology. 2021; 12: A.625386. https://doi.org/10.3389/fphar.2021.625386

2. Rud NK. Sampiev AM. Davitavyan NA. Main results of phytochemical and pharmacological research of Nigella sativa L. (review). Nauchnye vedomosti Belgorodskogo gosudarstvennogo universiteta. Seriya: Meditsina. Farmatsiya. 2013; 168(25): 207-212. (in Russ.).

3. Tiwari P. Jena S. Satpathy S. Sahu PK. Nigella sativa: Phytochemistry, Pharmacology and its Therapeutic Potential. Research J. Pharm. and Tech. 2019; 12(7): 3111-3116. doi.org/10.5958/0974-360X.2019.00526.2

4. Khazdair MR. Ghafari S. Sadeghi M. Possible therapeutic effects of Nigella sativa and its thymoquinone on COVID-19. Pharmaceutical Biology. 2021; 59(1): 696-703. doi.org/10.1080/13880209.2021.1931353

5. Kurkin VA. Mubinov AR. Avdeeva HV. Ryazanova TK. Comparative research of Fatty acid Composition and Volatile Components of Fatty oils from seeds of Nigella sativa and Arganiaspinosa. Research J. Pharm. and Tech. 2021; 14(3): 1586-1590. doi.org/10.5958/0974-360X.2021.00280.8

6. Aftab A. Yousaf Z. Javaid A. Rabbani A. Ahmed S. Khan F. Nigella sativa L. from traditional to contemporary medicine: a review. International Journal of Biology and Biotechnology. 2018; 15(2): 237-254.

7. Wijaya I. Ginting CN. Ginting SF. Ikhtiari R. Phytochemical and Antibacterial Analysis of the Formulated Cream of Black Cumin Honey. Research J. Pharm. and Tech. 2021; 14(11): 5691-5. doi.org/10.52711/0974-360X.2021.00989

8. Majumdar M. Samanta A. Roy A. Study of wound healing activity of different formulations of Nigella sativa seed extract. Research J. Pharm. and Tech. 2016; 9(12): 2097-2105. doi.org/10.5958/0974-360X.2016.00427.3

9. Mustafa NS. Mohamad N. Abu Bakar NH. Mohd Adnan LH. Jeharsae R. Talek M. Md. Fauzi NFA. Ahmad NZ. Neuroprotective effects of thymoquinone against MDMA-induced neurotoxicity. Research J. Pharm. and Tech. 2021; 14(11): 5945-2. doi.org/10.52711/0974-360X.2021.01033

10. Parveen A. Farooq MA. Kyunn WW. New Oleanane Type Saponin from the Aerial Parts of Nigella sativa with Anti-Oxidant and Anti-Diabetic Potential. Molecules. 2020; 25(9): A. 2171. doi.org/10.3390/molecules25092171.

11. Sharofova M.U. NuralievYu.N. Shabanov P.D. SagdievaSh.S. SuhrobovP.Sh. Numonov S.R. The study of the antidiabetic properties of the above-ground parts of black cumin (Nigella sativa L.). Problems of biological, medical and pharmaceutical chemistry. 2018; 21(10): 112−118. doi.org/10.29296/25877313-2018-10-21.

12. Tiwari P. Phenolics and Flavonoids and Antioxidant Potential of Balarishta Prepared by Traditional and Modern Methods. Asian J. Pharm. Ana. 2014; 4(1): 5-10.

13. Sutrisna E. Azizah T. Wahyuni S. Potency of Nigella sativa Linn. Seed as antidiabetic (preclinical study). Research Journal of Pharmacy and Technology. 2022; 15(1): 381-4. doi.org/10.52711/0974-360X.2022.00062

14. Rao KP. Kumari KS. Mohan S. Synthesis, Characterization and Antimicrobial activity of Some Flavones. Asian J. Research Chem. 2013; 6(2): 163-165.

15. Aftab A. Yousaf Z. Aftab Z. Younas A. Riaz N. Rashid M. Shamsheer HB. Razzaq Z. Javaid A. Pharmacological screening and GC-MS analysis of vegetative/reproductive parts of Nigella sativa L. Pakistan Journal of Pharmaceutical Sciences. 2020; 33(5): 2103-2111. doi.org/10.36721/PJPS.2020.33.5.REG.2103-2111.1.

16. Mabry TJ. Markham KR. Thomas MB. The Systematic Identification of Flavonoids. Springer Verlag, Berlin-Heidelberg-New York.; 1970; 354 p.

17. Saidin KS. Jais MR. Ismail EN. Ishak R. The effect of Nigella sativa and Eucheumacottonii in Collagen-induced Arthritis mice. Research J. Pharm. and Tech. 2020; 13(3): 1319-1323. doi.org/10.5958/0974-360X.2020.00243.7

18. Tiwari P. Patel RK. Estimation of Total Phenolics and Flavonoids and Antioxidant Potential of Ashwagandharishta Prepared by Traditional and Modern Methods. Asian J. Pharm. Ana. 2013; 3(4): 147-152.

Received on 20.06.2023 Modified on 03.11.2023

Research J. Pharm. and Tech. 2024; 17(3):1314-1319.

DOI: 10.52711/0974-360X.2024.00206