INTRODUCTION:

The hepatic is a vitally essential organ with several distinct functions of human body. It plays an important role of the central site for metabolism, synthesis and storage and discharge of exogenous and endogenous substances. It is associated with virtual growth of biochemical pathways and transformation of toxic excretion compounds.^1,2. So, it is exposure to a xenobiotic, medicinal agents and environmental pollutants and so on. Liver harm are related to twist of digestion ability and related with degradation of cells and addition in tissues in lipid peroxidation, utilization of decline glutathione levels. As well as the serum level of numerous biochemical markers such as basic phosphate, bilirubin, fatty oils, cholesterol, transaminases, triglycerides are increased the liver infection.^3,4,5.

In the recent study,an ancient herbal drugs used in the therapy of various liver disease. Furthermore, a vast number of herbal plants are initiate to show more hepatoprotective. Andrographis paniculata name called as Creat or green chireta, belong to the Acanthaceae family. Andrograhis paniculata belongs to plantae kingdom in order of lamiales containing genus of Andrographis with the species A.paniculata. Andrographis paniculata (Burm. f.) Nees is one of the widely distributed medicinal plants in India and using since ancient times in traditional ayurvedic systems of medicines^6,7. Andrographis paniculata (Burm. f.) Nees is one of the important herbs are used in various disease. Some of the herbal drugs and perfumery products used in the world are naturally in India. And the very beneficial and less side effects of products and easily available in the market. Andrographis paniculata has widely used in the treatment of disease including tuberculosis, fatty liver, constipation, cirrhosis, cough, hepatitis and so on^8,9,10.Andrographis paniculata has a widely used of pharmacological impacts and broadly used such as: anti-oxidant, antimalarial, anticancer, anti-inflammatory, antipyretic, hepatoprotective, anti-hiv, immunomodulatory, antifungal, antibacterial, antidiabetic, antifertility, larvicidal and ovicidal, anti-leishmanial^11,12. This plant has been reported on psychopharmacological activity. Different preclinical experiment reported the hepatoprotective effect of A.paniculata and its major constituent andrographolide^13.

Fig.1: Plant of Andrographis Paniculata

Chemistry of Andrographolide:

Andrographolide is a bioactive constituents found in parts of A. paniculata. The IUPAC name of andrographolide is 3α, 14,15, 18-tetrahydroxy-5β,9βH, 10α-labda-8,12-dien-16-oic acid γ-lactone and its common name are Kalmegh, Creat.^14,15,16. Molecular formula are C₂₀H₃₀O₅ and its weight 350.4. These are soluble in methanol, chloroform, acetone, ether and is not soluble in water^17.

Fig 2: Chemical Structure of Andrographolide

The bicyclic diterpenoid lactone is the commonly known chemical constituents found in Andrographis paniculata. The other constituents like andrographolide, andrograpin, neoandrographolide, panicolin and terpenoids or flavonoids, glycosides. The other constituents like Paniculide-C, Paniculide –B, Paniculide-A, 14-Deoxy-11-dehydroandrographolide, 5-Hydroxy-7,8,2-Trimethoxyflavone^18,19.

MATERIAL AND METHODS:

Phytochemistry: Chemical Required:

For experimental work the chemical reagent and solvents are procured from different companies i.e. CDH (Central Drug House), E. Merck India Ltd, FINAR, RenChem, Sigma Aldrich etc. These reagent and solvents are of LR grade. For TLC, Silica gel G (160-120 lattice) was gotten from Merck India Pvt. Ltd. Two solvent system for running the compounds on TLC plates are used as chloroform: methanol and used in column chromatography as a mobile phase. Methanol are used in extraction process of plant. And the bromo-benzaldehyde, Zncl₂and DMSO are used in preparation of semi-synthetic reaction.

Instrument Used:

The prepared compounds checked for melting point done by Lab India Melting point apparatus. The structure elucidation done through ¹H- NMR spectra were evaluated on the instrument of Bruker 400 Hz using Dimethyl sulphoxide (NMR graded6) and Chloroform as standard. And the Mass spectra were evaluated in molecular mass. The InfraRed spectra (IR) got from instrument Perkin Elmer FT-IR Spectrometer. For visualization of TLC spots Iodine glass chamber and UV-light and High-performance liquid chromatography (HPLC) were used.

Plant material:

The stems of Andrographis paniculata were collected from NIET greater Noida, Uttar Pradesh, India, in 2020.

Extraction of Andrographis paniculata plant: The extraction procedure stem of Andrographis paniculata kept dried for 10 days at room temperature. Further dried stem converted into powder form using mechanical grinder. The crushed powder was transfer into the round bottom flask in soxhlet apparatus. To the extract of drug with the help of methanol as a choice of solvent (1000ml) for 7 days by soxhlet apparatus. And the temperature is 30-70ºC. After the completion of extraction procedure proceeds for 72 hours for about 3 weeks. The extract was filter and the filtrate was evaporated with the help of rotary evaporator/ vacuum filtration. After the finishing of the extraction procedure rate were determined.

Rate yield = Extract value/powdered value *100

And the extract drug is treated with petroleum ether removal of excess fat of the drug. Then it is used for further experiment.

Isolation of andrographolide:

The column was packed with slurry of silica containing mesh size 100-200. Silica dissolved in chloroform and poured the column in wet packing in 50cm of column. The dried extract of A.paniculata herbal drug was dissolved in methanol and poured the column through the pipette at the top of prepared column for the collection of fractions. Different fractions are eluted with the ratio of chloroform: methanol solvent. The column was run with gradient of chloroform : methanol (90:10, 80:20, 70:30, 60:40, 50:50, 20:80, 10:90) finally 100% methanol and 8fractions (F1-F8) were collected. Band colour of fraction are different- different colour was formed with the different ratio of mobile phase. And the elute fractions was collected. After the elution fraction are evaporated at room temperature. Fraction F5 and F6, colourless crystals were isolated. The identity of crystals was confirmed by spectroscopic techniques. (Thin layer chromatography, U.V, I.R). After the completion of isolation process compound are used for further experiment process.

Preparation of semi- synthetic derivative:

In these preparation steps of Isolated Andrographolide derivative are prepared for using these procedure as follow: weigh the mixture of isolated andrographolide (0.5gm), and 4-bromo-benzaldehye (0.132gm), and ZnCl₂(80mg) was stirred in (1ml) DMSO at the room temperature for 5-6hours. After the completion of the reaction (checked by thin layer chromatography, Infrared spectroscopy, U.V, NMR spectroscopy). After the characterization reacted contents were diluted with the water and extracted with dichloromethane. The organic layer was dried over anhydrous Na₂SO₄and purified by column chromatography (chloroform: methanol) in the ratio of (99:1) to the afford of 3,19-(4-bromobenzylidene) Andrographolide.

Hepatoprotective activity: Paracetamol induced hepatoxicity model

Experimental design:

To study of the hepatoprotective activity the male wistar albino rat animal were taken from Central Animal House of NIET, Greater Noida. The project work provided with protocol number IAEC/ NIET / 2019 / 01 / 08 studies were performed in according to the guidance of CPCSEA registration^20. The tested animal was housed in polypropylene cages maintaining temperature 25 ± 20⁰C with proper twelve hour dark and light cycle. The animal kept with proper feed of water and food. Proper litter was removed within a week maintaining 23 echogenicity with maximum comfort provided to the animals. The experimental animals were divided into four grouped containing six in each and treated as follows:

Group -1 Control (Normal saline)

Group -2 Standard Liv-52 (2ml/kg),p.o

Group -3 Andrographolide (20mg/kg),p.o

Group -4 Semi-synthetic derivative of Andrographolide (20mg/kg), p.o

Table.1: Animal Data For Hepatoprotective Activity

S. No	Type	Albino Rats
1	Species	Wistar
2	Sex	Male
3	Age	2 months
4	Weight	150-250 gm
5	Number	24
6	Groups	4
7	Group I ( Normal saline)	Control (6)
8	Group II (Standard drug -2ml/kg)	Liv-52 (6)
9	Group III (Test 1-20mg/kg)	Andrographolide (6)
10	Group IV (Test 2- 20mg/kg)	Semi-synthethic derivative of Andrographolide- (6)

Fixation of dose: The animals were acclimatization for 21 days after bringing to the laboratory with everyday handling. The animals in each group follow:

Group -1 Control (Normal saline)

Group -2 Standard (Liv-52) 2mL/kg

Group -3 Andrographolide (20mg/kg)

Group -4 Semi-synthetic derivative (20mg/kg)

24 wistar albino rats (150-250) gm will be procured and the animals are divided into groups for hepatoprotective activity. For hepatoprotective activity rats will be fasted for 16 hours. Liv-52 refer to a hepatoprotective used as a standard drug (2ml/kg) orally. Normal saline was used as control to get ready arrangements of the concentrate and also the standard medication. Andrographolide and Semi-synthetic derivative (20 and 20mg/kg) were administered initially for 7 days. Hepatic damage was induced in rats by orally administered of paracetamol (500mg/kg) orally for seven days once in a day^21,22.

End of the study: After the completion of the treatment the rat will be sacrificed after the one night fasting and the animal will be sacrificed under ketamine hydrochloride anesthesia 24hours after the last dose. Blood were collected by cardiac puncture in plain tubes and liver were removed, rinsed in cold saline, blotted with filter paper and weighed.

Fig.3: Liver of male albino rat

Liver was collected and kept in 10%formalin solution for histopathological evaluation. And the serum chemical SGOT, SGPT and bilirubin levels were evaluated.

RESULTS:

Structure elucidation of the derivative:

The structures of andrographolide and its derivative are given in Fig.4. The physical and spectroscopic data of the isolated compound and the derivative were comparable with those reported in the literature.

Fig.4: Scheme of Andrographolide derivative

In-silico analysis:

In silico analysis showed that 3-19(4-bromo benzylidene) andrographolide had high log P value, which indicated low hydrophilicity which caused poor absorption. Calculated log S of derivative was also very low which indicated that it was comparatively moderated soluble than andrographolide.

Table.2: Computational study of physicochemical properties of andrographolide andits derivative

S. No	Properties	Andrographolide	3,19(4-bromobenzylidene) andrographolide derivative
1.	Molecular formula	C₂₀H₃₀O₅	C₂₇H₃₃BrO₅
2.	Molecular weight	350.45g/ mole	517.45g/mole
3.	Number of heavy atoms	25	33
4.	Number of rotatable bonds	3	3
5.	No. of H-bond acceptor	5	5
6.	No. of H-bond donor	3	1
7.	Log P	2.45	4.16
8.	Log S	-3.18	-6.00
9.	G.I absorption	High	High

Characterization of 3,19 (4-Bromo Benzylidene) Andrographolide Derivative:

Statistical Analysis:

The animals treated with andrographolide and its derivative showed reduction in the AST, ALT, and ALP and bilirubin levels was comparing with the normal control and standard (Liv-52) treated animals. Andrographolide derivative did not change the serum urea and creatinine levels.

The effect of andrographolide and semi-synthetic derivative on serum function biochemical parameters related to liver functions was reported as mean ± SEM of six animals in each groups.

Groups difference were evaluated by the help of ANOVA followed by Graph prism software and p≤ 0.05 were considered significant.

DISCUSSION:

In this study, we report the hepatoprotective effect of andrographolide and its derivative. Compounds having high logP and low logS values have low absorption and in this study, low log P and high log S values of 3, 19(4- bromo benzylidene andrographolide showed the availability of these compounds be higher than andrographolide^23,24. The evaluation of 3,19(4-bromo benzylidene )andrographolide derivative indicate the low irritability and high drug score than andrographolide. It is useful to reduce toxicity of andrographolide and improving the biological activity. Experimental study was also in according to the previous study. The increment in the% of AST, ALT and LDH by the treatment of andrographolide derivative due to hepatoprotective effect of the compound^25.

(A)

(B)

(C)

(D)

(E)

(F)

(G)

Fig.5: Histopathological evaluation of effect of liver in andrographolide and its derivative

Representations of microphotograph were present for (A) Normal control group, (B-E) standard (Liv-52), (F) Andrographolide (G) Semi- synthetic derivative of andrographolide.

CONCLUSION:

This study is an initial report on the effectiveness of 3, 19 (4-bromobenzylidene) andrographolide derivative for the treatment of hepatoprotective with comparatively high drug score, low irritability, improved efficacy on hepatic protection. However, detail evaluation on dose dependency, compare pharmacokinetics, molecular mechanism of action, safety of derivative and they are valid for treatment of hepatic disease in a lead molecule.

ACKNOWLEDGEMENT:

The authors would like to thanks Dr. Avijit Majumdar, Director Pharmacy Institute NIET, Dr. Salahuddin, H.O.D Pharmaceutical Department of Chemistry, Mr. Rajnish Shukla Assistant Professor, Pharmacy Institute NIET, Greater Noida, Uttar Pradesh.

CONFLICT OF INTEREST:

The author has declared no conflict of interest.

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Received on 22.01.2021 Modified on 26.07.2021

Research J. Pharm. and Tech 2022; 15(9):4055-4060.

DOI: 10.52711/0974-360X.2022.00680

S. No	Groups	ALT	AST	ALP	Albumin	Creatinine	Urea
1.	Normal control group	90.86± 13.70	40.16±6.99	12.14±5.82	4.15±0.20	0.070±0.03	18.20±2.72
2.	Standard group (Liv-52)	113.17±16.60	186.17±12.95	165.83±11.24	4.17±0.19	0.071±0.04	19.22±2.34
3.	Test compound Andrographolide	141.0±50.29	61.95±17.46	28.37±10.95	3.56±0.50	0.071±0.07	21.38±1.35
4.	3,19(4-bromo benzylidene) andrographolide derivative	145.0±52.27	63.97±19.47	30.39±12.96	4.57±0.52	0.073±0.09	22.39±2.37