Novel “HYDALJSS08” Polyherbal Formulation Development and Ultra-Performance Liquid Chromatographic Separation, Estimation of Berberine in Cissampelos pareira Roots, Tinospora cordifolia Stems, and A Marketed Siddha-Based Polyherbal Formulation “Kabusura Kudineer.”

 

Ramkishan Jatoth¹*, Dhanabal S.P ¹, Duraiswamy Basavan¹, V. Senthil²,

T. Ganesh³, Jeyprakash. M.R⁴

¹Department of Pharmacognosy and Phytopharmacy, JSS College of Pharmacy,

JSS Academy of Higher Education and Research, Ooty, Nilgiris, Tamilnadu, India.

²Department of Pharmaceutics, J.K.K. Nattraja College of Pharmacy, Komarapalayam, Erode, Tamilnadu, India.

³Manager- R&D (Research & Development), SKM Siddha and Ayurveda (India) Pvt Ltd, Tamilnadu, India.

⁵Dept of Pharmaceutical Analysis, JSS College of Pharmacy,

JSS Academy of Higher Education and Research, Ooty, Nilgiris, Tamilnadu, India.

 

ABSTRACT:

The present study aims to develop and document an analytical method for Berberine in the chloroform fraction of Cissampelos pareira (Menispermaceae)roots, Tinospora cordifolia (Menispermaceae) stems, and Polyherbal formulations (Marketed & Developed). During the pandemic Covid-19 ministry of Ayush, Govt of India was recommended a Siddha-based polyherbal formulation known as “Kabusura Kudineer” for immune booster and treatment of Covid -19. False pareira roots having active biomarker substances are BBE proved for its activity of Anti-viral, and immunomodulatory. Preliminary identification of Berberine in chloroform fraction of Cissampelos pareira roots, Tinospora cordifolia stems was done by the TLC, mobile phase used as a Toluene: Acetone: Water (5:15:1) and Chloroform: methanol (70:20). The UPLC was performed on a column, X Bridge BEH C18 Column, 130Å, 3.5µm, 4.6mm X 150mm, 1/pk and Solvent system were 0.1% Trifluoroacetic acid: Methanol (60:40). The effluents were observed at 272nm in UV detector. The Rf value of Berberine, Chloroform fraction of Cissampelos pareira roots was 0.23, and the Tinospora cordifolia stems was 0.63. The valid UPLC method parameter for BBE is system precision, SD (5433.07),  the Regression equation was found y = 20570 x−182430, Correlation coefficient (R2) 0.9993. The adequate Linearity concentration was found to be 50 to 150µg/ml, LODs (4.02µg/ml), LOQs (12.17µg/ml), Method precision for intraday, Interday %RSD (0.7), (1.4), SD (16064.5), (32811.5), Recovery 98%, and 102%. BBE content was found in a formulation (“KabusuraKudineer” churna- 20.9360µg/ml, Developed Hydroalcoholic Polyherbal formulation - 21.4370µg/ml), and the Chloroform fraction of Cissampelos Pareira roots (CHFCP), Chloroform fraction of Tinospora cordifolia stems (CHFTC) was 28.9090µg/ml and 73.2050µg/ml. The developed liquid chromatography (UPLC) methods have enabled simple, rapid, easy, accurate, reproducible, and linear analysis of BBE in Chloroform fraction of velvet roots, Tinospora cordifolia stems, and Ayurvedic, Siddha -based Polyherbal formulations.

 

 

 

 

INTRODUCTION: 

Siddha based polyherbal formulation known as “Kabusura Kudineer”. It is containing fifteen plant crude materials; they are used as an immune booster. It is manufactured by combining all dried crude drugs in equal proportion and pulverized. During the pandemic time of the Covid-19 ministry of Ayush, Govt of India was recommended the Siddha-based polyherbal formulation “Kabusura Kudineer” for immune booster and treatment of Covid-19. The composition of  Kabusura Kudineer and newly developed “Hydaljss08” Polyherbal formulation is Andrographis paniculata whole plant (Acanthaceae), Zingiber officinalis rhizomes (Zingiberaceae), Piper longum dried fruits (Piperaceae), Terminalia chebula dried fruits (Combretaceae), Syzygium aromaticum flower buds (Myrtaceae), Tragia involucrata roots (Euphorbiaceae), Adhatoda vesica leaf (Acanthaceae) Tinospora cordifolia stem (Menispermaceae), Anacyclus pyrethrum roots (Asteraceae), Hygrophila auriculata whole plant (Acanthaceae), Coleus amboinicus root- Lamiaceae, Saussurea lappa roots- Asteraceae, Clerodendrum serratum roots- Verbenaceae, Cissampelos pareira roots (Menispermaceae), Cyperus rotundus roots (Cyperaceae). The active compounds responsible for Anti-viral and immunomodulatory activity are 6-gingerol, zingerone, eugenol, biflorin, eugenin, piperine, pellitorine, chebulinic acid, chebulagic acid, gallic acid, ellagic acid, corilagin, quercetin, inulin, lupeol, beta-sitosterol, betulin, vasicine, vasicinone, apigenin, luteolin, rutin, Costunolide, Dehydrocostus lactone, berberine, cordifolioside, syringin, and andrographolide. The above compounds are responsible for anti-viral and immunomodulatory activity may be due to their synergistic effects. One of the active compounds of Cissampelos pareira roots and Tinospora cordifolia stems is BBE.¹ The principal objective of this work was to develop and document an analytical technique for Berberine by using TLC, a liquid chromatography method for identification and quantification of the chemical markers of marketed Siddha-based polyherbal formulations known as “Kabusura kudineer”, Newly developed “Hydaljss08” Polyherbal formulation (Asava/ Arishta), and in Chloroform fraction of Cissampelos Pareira roots (CHFCP), and Chloroform fraction of Tinospora cordifolia stems (CHFTC).

 

Covid-19 is a contagious disease, also known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), people have believed it originated the bats and was transmitted to humans.^2,3 The first reported case of respiratory illness was in Wuhan City, Hubei Province, China.⁴ The disease has since spread worldwide, leading to an ongoing pandemic. The official names Covid-19 and SARS-CoV-2 were issued by the World Health Organization on 11 February 2020.^5,6,7Globally, as of 4:36pm CET, 26^th July 2023, there have been 76, 85, 60, 727confirmed cases of Covid-19, including 69, 52, 522 deaths, reported to WHO. As of 4 November 2022, a total of 13, 47, 43, 48, 801 vaccine doses have been administered.⁸

Cissampelos pareira Linn. var. hirsuta belongs to the family Menispermaceae. It is commonly known as venivel. The plant is found throughout the tropical and sub-tropical areas of India and Ceylon⁹. The roots of Cissampelos pareira (Patha) in ethnomedicinal practices are used in the treatment of various ailments related to urinary problems and skin infections, and in tumor inhibitor activity, antibacterial, antimalarial, diuretic activity, anticonvulsant activity, etc.¹⁰ The biologically active chemical constituents of Cissampelos pareira roots are flavone, alkaloids like Berberine, cissampareine, cissamine, pareirubrine A and B.¹¹  It also contains pareitropone, trandrine, hayatin, and beta-sitosterol.¹² The phytochemical and biological investigation of Cissampelos pareira roots leads to the isolation of seven isoquinoline alkaloids along with six knowns and one new, they are namely magnoflorine, magnocurarine, cissamine, curine, hayatinine, and cycleanine.¹³ The roots of the Barbasco Linn. during the process of phytochemical analysis lead to the isolation of a new pyrrole alkaloid, known as “cissampeline”, along with other well-known basic substances called coclaurine, tetrandrine, curine, cyclanoline, berberine, homoaromoline, nor-N׳-chondrocurine, and trans-N-feruloyl tyramine.¹⁴

 

Tinospora cordifolia (Menispermaceae) (Wild.) Hook. f. and Thoms. (Guduchi) is a glabrous, large, deciduous climbing shrub. It spreads throughout the tropical Indian subcontinent and China, growing to an altitude of 300 m. In Hindi, the plant is commonly known as Giloe, which is a Hindu mythological term that refers to the heavenly elixir that has saved celestial beings from old age and kept them eternally.¹⁵ Phytochemical screening of Tinospora cordifolia stems contains glycosides, alkaloids, steroids, sesquiterpenoids, aliphatic compounds, essential oils, a mixture of fatty acids, and polysaccharides. A Phytochemical investigation of “Tinospora cordifolia” stems have been isolated different chemical active components, they are tinosporide, cordifolide tinosporine, tinosporaside, cordifol, heptacosanol, clerodane furano diterpene, diterpenoid furanolactone tinosporidine, columbin, and β-sitosterol. The leaves of the “Tinospora cordifolia” plants are rich in proteins about 11.2% and are rich in calcium and phosphorus. The major active groups of chemical compounds present in plants are protoberberine alkaloids, polysaccharides, glycosides terpenoids, and glycosides terpenoids.¹⁶ Guduchi plant stems also contain, active compounds Berberine, magnoflorine, palmatine, columbamine, isocolumbin, syringin, tinocardioside, cordiol, palmatrubine, palmatine, jatrorrhizine, columbamine, cycloeuphordenol, 1-cyclohexyl-11-heneicosanone,2-Hydroxy-4- methoxy benzaldehyde.¹⁷

 

MATERIALS AND METHODS:

Raw materials:

The authenticated false pareira roots (Cissampelos pareira) and Tinospora  cordifolia stems purchased from the KRC crude drugs Chennai (13.0827°N, 80.2707°E). Purchased materials dried at 40^◦C using a hot air oven. Dried false pareira root materials pulverized by using a grinder as a fine powder (10mesh outlet sieve) and packed in a dry airtight container till further use.¹⁸

 

Extraction and Fractionation of Berberine from Cissampelos pareira Roots:

Cissampelos pareira dried roots fine powder (500g) transferred in a 2000ml round bottom flask and extracted with 1500ml of demineralized water for one week. The extract was filtered and collected filtrate and evaporated with a rota evaporator to get viscous mass (123gm). The crude extract was acidified with 1N HCL and then treated with 1N Sodium hydroxide. The obtained ppt was extracted with diethyl ether and that fraction was concentrated. The Aqueous layer was further extracted with chloroform. The extract was repeated until the last fraction did not give any precipitate with Dragendorff’s reagent. The collected chloroform fraction concentrated it gives yellow colour mass/ crystals.¹⁹ Preliminary phytochemical identifications of CHFCP were identified by TLC (pre-coated) mobile phase and was used as a Toluene: Acetone: Water (5:15:1). The Rf value was found to be 0.23. Eluted spot detected by UV chamber as detecting agent.

 

Extraction and Fractionation of Berberine from Tinospora cordifolia Stems:

500 gm of dried Tinospora cordifolia stems powder was transferred in a 2000ml round bottom flask and macerated with ethanol at room temperature. Filter the extract and collect the filtrate. Evaporate the filtrate and dried it (10gm). The dried extract is washed with petroleum ether and suspended in water. The suspended extract is partitioned with butanol, hexane, chloroform, ethyl acetate, and toluene and collected in each fraction, and evaporated in the solvent. The collected chloroform and butanol fraction concentrated it give yellow colour mass/crystals. Preliminary phytochemical identification of CHFTP was identified by TLC (precoated) mobile phase was used as a Chloroform: Methanol (70:20). The Rf value was found to be 0.63. Eluted spots were detected by the iodine as a detecting agent and the naked eye.²⁰ The results data were summarized Fig. 1, 2 and 3. FT-IR Spectrum was taken, and functional groups were identified for CHFTC.

 

             Fig. 1: CHFTC                            Fig.2: CHFCP

 

Fig 1, 2. TLC of Berberine in Chloroform fraction of Tinospora cordifoliastems, Chloroform fraction of Cissampelos pareira roots and Formulations.

 

Chemical marker and Chemicals:

The Berberine reference standard was purchased from “Yucca enterprises, Mumbai” (19.0206° N, 72.8679° E). Require solvents known as methanol, ethanol, acetone, toluene, ethyl acetate, chloroform, and hexane purchased from the Central drug house (P) Ltd, Gujarat, and chemicals collected from the Dept. of Pharmacognosy and Pharmaceutical Chemistry, JSSAHER-JSS College of Pharmacy, Ooty, Tamil Nadu, India. (Longitude -76°42′25.56′′E (76.7071), Latitude-11°24′4.07′′N (11.401127), and Chandra labs, Hyderabad, Telangana, India (Longitude-78.4271639, Latitude- 17.4760781.

 

HPLC System and Instruments:

The liquid chromatography was discharged on a Shimadzu LC2010 HPLC (UPLC) instrument composed of an ultrafast autosampler, and a UV-VIS detector. The LC-2010 is also designed for ease of use by automating the analysis process LAB Solution software. X Bridge BEH C18 Column, 130Å, 3.5µm, 4.6mm X 150mm, 1/pkcolumn was used as stationary phase.²¹ FT-IR Model-8400S (Shimadzu), KBR pressing, Software-Shimadzu-IR-Solution.

 

Preparation of Standard and Sample Solutions:

Standard preparation and Sample Preparations:

50mg of weighed Berberine was weighed, transferred into a 100ml volumetric flask, diluted with the solvent system then make up to the mark with the solvent. The final concentration was made up to 100µg/ with the mobile phase.²²

 

“Hydaljss08” Polyherbal Formulation Preparations:

Accurately taken 5ml of Liquid sample and transferred into 50ml volumetric flask, added 30ml of diluent and sonicated for 10min, made final volume with diluent, and mixed well. Centrifuged this solution at 5000rpm for 10min. The supernatant was further diluted from 1ml to 10ml with diluent, mixed well, and injected into a liquid chromatography (UPLC) system.²³ The prepared solution is stored under refrigerator conditions.

 

METHOD VALIDATION:

Documentation of the analytical strategy was done according to the ICH guidelines. The method was documented for Accuracy, specificity, robustness, linearity, precision, repeatability, reproducibility, system suitability, the limit of quantitation (LOQ), and the limit of detection (LOD). All the validation parameter data of Berberine was illustrated in Table-1-3 and Fig.3.²⁴

 

Fig. 3: Structure of Berberine.

 

RESULTS AND DISCUSSION:

The recommended method was found to be simple, novel, sensitive, accurate, precise, economical, rapid for the regimen for the liquid chromatography development, documentation of Berberine in a marketed Siddha-based polyherbal formulation known as “Kabusura Kudineer”, Developed “Hydaljss08” Polyherbal formulation, Chloroform fraction Cissampelos pareira roots (CHFCP), and  in a chloroform fraction of Tinospora cordifolia stems (CHCFTC).^25,26 The preliminary identification of BBE by thin layer chromatography, mobile phase used as a Toluene: Acetone: water (5:15:1) in CHFCP, and CHFTC (Chloroform: Methanol (70:20), and Polyherbal formulation known as “Kabusura Kudineer” (Marketed) result data summarized in Fig 1,2, the Rf value of BBE was found to be 0.23 (CHFCP), 0.63 (CHFTC) respectively. HPLC development and validation of standard BBE were executed along with all the samples. The optimized mobile phase for liquid chromatography development was used as a 0.1% Trifluoroacetic acid: Methanol (60:40) and Standard BBE was detected at 272nm in a UV detector. The retention time of BBE is found as 3.4 min and the full run time of the chromatogram was 10 min. To determine the System suitability of BBE 100µg/ml sample was injected six times to analyse accurate, precise results, and the chromatograms were recorded. Standard deviation (5433.07) and %RSD (0.2) were found as adequate. The plate count and tailing factor results were found to be within the limits and the %RSD was found to be 0.266% so the system is suitable and gives precise results, data are summarized in Table 1 and Fig 4. A calibration curve was established for BBE by injecting 50, 75, 100, 125, and 150µg/ml of marker solution. For analysis, the average peak area of BBE linearity was determined, by injecting different concentrations of standard, and then, the regression equation was found to be y = 20570 x + 182430 and the correlation coefficient (R2) of BBE was 0.9993 respectively. Thus, adequate linearity was observed when the BBE concentration ranged from 50 to 150 µg/ml, data results are summarized in Table 1, 2 and Fig 5.

 

Table 1: Intra, inter-day Results of Method Precision, Results of Linearity and Range of Berberine (BBE).

S. No	Intra-day Precision (n=6)		Inter-day precision (n=6)		Linearity and Range
	Con. 100 µg /ml	Area	Con. 100 µg /ml	Area	Concentration (µg/mL)	Area
1	1	2281838	1	2277007	50.0	1206568
2	2	2249309	2	2297077	75.0	1706287
3	3	2252615	3	2260200	100.0	2270157
4	4	2266459	4	2353079	125.0	2766172
5	5	2257948	5	2304786	150.0	3247846
6	6	2288734	6	2321244	-	-
7	Average	2266151	Average	2302232	-	-
8	Std dev	16064.5	Std dev	32811.5	-	-
9	% RSD	0.7	% RSD	1.4	-	-

 

Table 2: System Suitability and Ruggedness results of Berberine (BBE).

S. No	Concentration (µg/mL)	Area	Ruggedness Results of BBE.
1	Linearity and Range	50-150 µg/ml	Berberine	%Assay
2	Regression equation	y = 20570 x + 182430	Analyst 01	99.6%
3	Correlation coefficient	0.9993	Analyst 02	100.1 %
4	Theoretical plates	14550.75	%RSD	0.4%
5	Tailing Factor	1.295	-	-
6	LOD	4.02	-	-
7	LOQ	12.17	-	-
8	Slope	20570	-	-
9	Intercept	182430	-	-

 

Fig. 4: Typical UPLC Chromatogram of Std Berberine.

 

 

Fig 5: Typical Linearity Graph of Berberine (BBE).

 

Table 3: Results of Recovery Studies for Berberine (BBE).

S. No	Concentration in (%)	Concentration in (µg/ml)	Area	% Recovered	% Recovery
1	50	50	1173420	101.3	101.5
		50	1176413	101.2
2		50	1156572	101.9
	100	100	2292700	101.0	100.4
		100	2282319	100.5
		100	2267610	99.9
3	150	150	3323734	98.2	98.5
		150	3283963	98.4
		150	3260249	99.0

 

Table 4: Percentage Purity Calculation of Berberine in Fraction of Cissampelos pareira Root, Tinospora cordifolia stems and Polyherbal Formulations.

S. No	Sample	Area	% Assay	in µg/ml	in mg/ml
1	Berberine Reference Standard.	1173420	Label Claims 97.4%.
2	Estimation of BBE inChloroform fraction of Cissampelos Pareira roots	656490	28.9	0.0289	28.9090
3	Estimation of BBE inChloroform fraction of Tinospora cordifolia stems	1662388	73.2	0.0732	73.2050
4	Estimation of BBE in Developed “Hydaljss08” Polyherbal Formulations	486807	21.4	0.0214	21.4370
	Estimation of BBE in “Kabusura Kudineer” Marketed.	475438	20.9	0.0209	20.9360

 

 

Fig 6: Typical UPLC Chromatogram of BBE in CHFTC Stems.

 

Fig 7. Typical UPLC Chromatogram of BBE in “Kabusura Kudineer.” Marketed.

 

Fig 8: Typical UPLC Chromatogram of BBE in “Hydaljss08” Polyherbal Formulation.

 

 

The LODs and LOQs for Berberine were found to be 4.02µg/ml and 12.17µg/ml respectively, as summarized in Table 2. Method precision was analysed by six different concentrations Intraday, Interday % of RSD (0.7), (1.4), and the standard deviation (16064.5), (32811.5) of samples for BBE was found to be within the acceptance criteria (less than 2.0%). Hence the method is precise data results are summarized in Table 1.

 

The recovery studies were carried out three times and the percentage recovery and percentage mean recovery were calculated for drugs. The percentage mean recovery of BBE was found between 98% and 102%, and data results are summarized in Table 3. The Robustness of the sample was determined. The obtained results are deliberate by variations in method parameters was summarized. The tailing factor and theoretical plates of the sample were found to be within the limits of small variations of flow rate and wavelength, data were summarized in Table 2. The ruggedness of the method was studied by determining the different types of variation in sample analysis by performing the Assay by two different analysts or two different systems etc. From the results % Assay and %, RSD obtained acceptance criteria of 2.0% so the method is rugged, data results are summarized in Table 2. In specificity, the study observed that diluent or excipient peaks do not interfere with the BBE peak. Estimation of BBE in CHFCP, CHFTC, and marketed Siddha-based polyherbal formulation known as “Kabusura Kudineer,” a Newly developed “Hydaljss08” Polyherbal formulation was found satisfactory. All the Sample results data as summarized in Table 4 and Fig6 - 8.^27,28,29

 

CONCLUSION:

The present developed method a rapid, simple, accurate, and reliable method was developed to estimation for the chemical marker present in chloroform fraction Cissampelos pareira roots, Tinospora cordifolia stem, and marketed Siddha-based polyherbal formulation known as “Kabusura Kudineer”, Newly developed “Hydaljss08” Polyherbal formulation by using liquid chromatography.³⁰ The obtained results revealed that the method showed better linearity, recovery, reproducibility, and low limits of detection and quantification. Thus, it is acceptable to be used in the quantification of berberine in Ayurvedic polyherbal formulations chloroform fraction of Cissampelos pareira roots, and Tinospora cordifolia stems.^31,32.

 

ACKNOWLEDGEMENTS:

The authors acknowledge “Yucca enterprises”, Mumbai, India for providing reference standard Berberine, and Chandra Labs, Hyderabad for Providing Laboratories Analysis facility. They also acknowledge KRC Crude Drugs Chennai, Tamilnadu, India, for providing Tinospora cordifolia stems, Cissampelos pareira roots dried fruits, and JSSAHER-JSS College of Pharmacy, Ooty, Tamilnadu, India, for providing of the Laboratories facility.

 

ABBREVIATIONS:

UPLC: Ultra-performance liquid chromatography; SARS-COV-2: severe acute respiratory syndrome coronavirus  2; WHO: World Health Organization; HPLC: High-performance liquid chromatography; SD: Standard deviation; RSD: Relative standard deviation; LOD: Limit of detection; LOQ: Limit of quantification; ICH: International Council for Harmonisation, BBE: Berberine, CHFCP-Chloroform fraction of Cissampelos pareira roots, CHFTC- Chloroform fraction of Tinospora cordifolia stems, CET-Central European Time.

 

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32.   Flávia Bonfim Lima et al. Identification and Quantification by HPLC DAD of Furosemide as a Co-adulterant in Products of Natural Origin; Journal of Natural Remedies. 2019; 20(2): 166-172. DOI: 10.18311/jnr/2020/24396.

 

 

 

Accepted on 28.09.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(11):5114-5120.