Hepatoprotective and Antioxidant Capacity of Clerodendrum paniculatum Flower Extracts against Carbon Tetrachloride-Induced Hepatotoxicity in Rats

Abstract

The aim of the presented work involves the isolation, characterization, and evaluation of hepatoprotective potential of Clerodendrum paniculatum flower extracts. For this purpose, petroleum ether, chloroform, ethyl acetate, alcohol, and water extracts of C. paniculatum flower were screened for the flavonoid and phenolic content and quantified. Various antioxidant activity assays including 2,2′-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO) radical scavenging, 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and reducing ability were carried out. Of the above methods, the alcoholic extract exhibited high antioxidant potential and was selected further for the hepatoprotective evaluations. Hepatoprotective evaluation of the alcoholic extract was carried out for carbon tetrachloride (CCl₄)-intoxicated model systems. Enzymes associated with liver functions were estimated, and histopathological evaluations were carried out to monitor the liver architecture. Prominently, reduced levels of various associated enzymes along with increased protein content were observed when the liver specimen was pretreated with the extract. Moreover, the liver architecture was almost comparable to that of the normal control group. The column chromatographic analysis of the extract revealed 13 fractions to possess high phenolics and flavonoid contents. The best two fractions were identified for in vitro hepatoprotective evaluation in the goat liver model. Furthermore, the GC–MS analyses of the fractions were carried out followed by a library search, to identify the constituents responsible for the hepatoprotective activity which revealed the presence of four major constituents—pilocarpine, glyceric acid, pangamic acid, and gallic acid. An in vitro hepatoprotective study of the isolated fractions showed better activity compared to the whole alcoholic extract, and the results were comparable to the normal group taken as a control. The investigations with an in vitro model suggest that the isolated fraction with rich flavonoid content showed better hepatoprotective activity. GC–MS analysis of the fractions that displayed good hepatoprotective activity suggested the presence of pilocarpine, glyceric acid, pangamic acid, and gallic acid, while HPTLC analysis revealed the presence of quercetin.

Introduction

Medicinal plants are known to possess several primary and secondary metabolites. The secondary metabolites in particular are involved in many biological activities. They are also effective against managing oxidative stress and hence responsible to fight against various diseases caused by stress.^1,2 The diverse chemical structures of such secondary metabolites have significant contributions toward new drug development processes. It has been observed in the past several decades that natural resources provide an enhanced biological activity, while the demands for the development of similar synthetic lead compound are limited.³ Antioxidants have the capacity to scavenge the free radicals, which are generated by metabolism and to some extent by various diseases.⁴ The endogenous antioxidant system itself may be able to fight against the deleterious effects produced by oxidative stress. This includes the enzymes such as catalase, superoxide dismutase, glutathione,and so on that can scavenge free radicals and protect biological systems to some extent. The endogenous antioxidant mechanism reinforcement or external antioxidant supplements may help overcome the situation.⁵

Genus Clerodendrum belonging to family Verbenaceae or Lamiaceae has more than 500 species. This genus is distributed in herbs and small trees. Clerodendrum species are reported to possess anti-inflammatory, anti-diabetic, anti-cancer, anti-malarial properties, and so on. They comprise various classes of constituents like flavonoids, phenolics, terpenes, steroids, volatile constituents, and so on.⁶ Natural sources are more widely promoted and recommended for they display minimal side effects, as compared to synthetic agents.⁷ Drug discovery from natural resources is highly challenging as the processes like authentication, screening, isolation, structural elucidation, and so on. require expertise and well-experienced support. On the other hand, there has always been a huge demand on these natural sources for their excellent safety parameters.⁸Clerodendrum paniculatum (C. paniculatum) leaves, roots, and so on. are reported for their anti-oxidant, anti-inflammatory, hepatoprotective, and anti-diabetic activities and so on. However, the flower counterpart of this plant has not been investigated for any of the medicinal activities including their hepatoprotective roles in animal models. Herein, we have evaluated the hepatoprotective role of the alcoholic extract of the flower part of C. paniculatum against CCl₄-induced hepatotoxicity in female Wistar Albino rats and justified its pharmacological implications toward its use as traditional medicine.

Results

The phytochemical screening of the flower extract indicated the presence of carbohydrates, tannins, phenolics, flavonoids, proteins, and steroids, and the results are depicted in Table 1. The presence of phenolics and flavonoids contents is considered as the main indicators of antioxidant activity in herbals.

Table 1. Phytochemical Screening for the Flower Extract of C. paniculatum.

chemical class	petroleum ether	chloroform	ethyl acetate	alcohola	watera
proteins	-	-	-	-	++
carbohydrates	-	+	+	++	+++
flavonoids	-	-	-	+++	++
tannins and phenolics	-	-	-	+++	++
steroids	+	+	+	-	-
glycosides	-	-	-	-	-
alkaloids	-	-	-	-	-
volatile oils	-	-	-	-	-

^a+++: highly present, ++: moderately present, +: low, and -: absent.

The total phenolic content,⁹ as quantified by Folin-Ciocalteu’s method, indicates the highest activity (378.5 ± 0.883 mg QE/g) from the alcoholic extract, as compared to other extracts. The alcoholic extract of C. paniculatum flower showed the highest flavonoid content and hence high free-radical scavenging property (393.23 ± 1.23 mg GAW/g) (Table 2) compared to other extracts, as evaluated by the AlCl₃ colorimetric method.

Table 2. Quantification of Total Phenolics and Flavonoids in Extracts of C. paniculatum Flower.

sample	total phenolic content (mg GAE/g)	flavonoid content (mg QE/g)
petroleum ether	81.5 ± 0.91	64.03 ± 1.21
chloroform	114.6 ± 0.46	137.10 ± 1.85
ethyl acetate	148.6 ± 0.81	121.27 ± 1.20
alcohol	378.5 ± 0.88	393.23 ± 1.33
water	108.9 ± 1.15	131.07 ± 0.96

Antioxidant activity of the extracts was evaluated by four different methods.¹⁰ Antioxidants have a high impact on diseases as they can scavenge the free radicals and thereby modify or prevent the diseases.¹¹ DPPH assay carried out with the alcoholic extract shows better activity with IC₅₀ of 62.28 ± 0.51 μg/mL, and the values are presented in Table 3 and Figure S1 (Supporting Information).

Table 3. Antioxidant Potential, as Determined by DPPH Assay with the Alcoholic Extract of C. paniculatum Flower.

Sl. no.	sample	IC50 (μg/mL) (mean ± S.D)
1	standard (ascorbic acid)	48.74 ± 0.21
2	petroleum ether	693.59 ± 13.51a
3	chloroform	409.17 ± 2.16a
4	ethyl acetate	568.54 ± 5.27a
5	alcohol	62.28 ± 0.51a
6	water	342.07 ± 0.95a

^ap < 0.01, as compared with the standard group.

DPPH is widely used as an evaluation technique due to its ease of reaction. In the ABTS assay method, radical scavenging ability of the extracts was evaluated using a ABTS++ modified solution by the reaction between ABTS and potassium persulfate solution,¹² and the IC₅₀ of the extracts was calculated. The alcoholic extract presented the IC₅₀ value as 2362.71 ± 9.39 μg/mL, while the IC₅₀ value of the standard ascorbic acid was evaluated as 941.09 ± 8.312 μg/mL [Table 4 and Figure S2 (Supporting Information)].

Table 4. Antioxidant Activity, as Evaluated by ABTS Assay with the Alcoholic Extract of C. paniculatum Flower.

Sl. no.	sample	IC50 (μg/mL) (mean ± S.D)
1	standard (ascorbic acid)	941.09 ± 8.31
2	petroleum ether	7986 ± 2.00a
3	chloroform	3643.36 ± 1.37a
4	ethyl acetate	8628.2 ± 6.21a
5	alcohol	2362.71 ± 9.39a
6	water	8781.6 ± 16.31a

^ap < 0.01, as compared with the standard group.

Nitric oxide radical scavenging activity results of the extract were evaluated by comparing with standard gallic acid. Nitric oxide is produced by the reaction between aqueous sodium nitroprusside, oxygen, and nitrite ions, under physiological pH conditions.¹³ Antioxidants help scavenge this free radical, and this property was compared with the standard. In this method also, the alcoholic extract showed better activity than all other extracts with an IC₅₀ value of 193.09 ± 5.84 μg/mL, and the results are comparable to the standard (147.11 ± 10.20 μg/mL); the results are depicted in Table 5 and Figure S3 (Supporting Information).

Table 5. Antioxidant Activity, as Determined by Nitric Oxide Radical Scavenging Assay with the Alcoholic Extract of C. paniculatum Flower.

Sl. no.	sample	IC50 (μg/mL) (mean ± S.D)
1	standard (gallic acid)	147.11 ± 10.20
2	petroleum ether	4765.42 ± 6.88a
3	chloroform	1129.17 ± 17.74a
4	ethyl acetate	3970.74 ± 3.57a
5	alcohol	193.09 ± 5.84a
6	water	1384.17 ± 13.77a

^ap < 0.01, as compared with the standard group.

Reducing power ability of the extracts was determined using potassium ferricyanide. The ability of the extract to reduce potassium ferricyanide (Fe III) to potassium ferrocyanide (Fe II) was measured by the reaction with trichloroacetic acid and FeCl₃.¹⁴ All the extracts exhibited a dose-dependent response. Quercetin was used as the standard, and its response was observed between 1.05 ± 0.001 and 2.44 ± 0.0005 μg/mL. A significant increase in absorbance indicates a better reducing ability, and the same was observed as the alcoholic extract of C. paniculatum flower (Figure 1).

Figure 1.

Determination of reducing power for various solvent extracts from the flowers of C. paniculatum, where CPFP denotes the C. paniculatum flower petroleum ether extract, CPFC indicates the C. paniculatum flower chloroform extract, CPFE indicates the C. paniculatum flower ethyl acetate extract, CPFA indicates the C. paniculatum flower alcohol extract, and CPFW indicates the C. paniculatum flower water extract.

An acute oral toxicity study of the alcoholic extract at a concentration of 2000 mg/kg b.w. revealed that the extract is safe up to the abovementioned concentration range. Hepatoprotective investigation was carried out with the CCl₄ intoxicated model. The liver enzymes and protein levels were checked for all groups of animals, and the results are depicted in Table 6 and Figure S4 (Supporting Information).

Table 6. SGOT, SGPT, ALP, Total and Direct Bilirubin, and Total Protein Levels of All Groups of Animals^a,b.

				Bilirubin (mg/dL)
groups	SGOT (IU/L)	SGPT (IU/L)	ALP (IU/L)	total	direct	total protein (g/dL)
vehicle control	59.33 ± 2.55**	52.83 ± 2.33**	101.5 ± 7.92**	0.703 ± 0.02**	0.163 ± 0.01**	8.47 ± 0.02**
toxic control	145.83 ± 4.45	112.17 ± 3.05	324.17 ± 3.55	1.42 ± 0.05	0.34 ± 0.02	6.28 ± 0.03
standard (silymarin)	66.33 ± 2.19**	56.67 ± 2.20**	109.83 ± 3.21**	0.752 ± 0.01**	0.17 ± 0.01**	8.36 ± 0.02**
CPFA (200 mg/kg)	64.67 ± 2.08**	62.83 ± 0.79**	128.50 ± 2.88**	0.787 ± 0.03**	0.18 ± 0.01**	8.12 ± 0.01**
CPFA (400 mg/kg)	59.83 ± 1.70**	58.67 ± 2.63**	120.33 ± 2.63**	0.638 ± 0.03**	0.14 ± 0.01**	8.19 ± 0.02**

^aValues are expressed as mean ± SEM of six rats in each group. **p < 0.05, as compared with the toxic control group.

^bCPFA: C. paniculatum flower alcoholic extract, SGOT: serum glutamate oxaloacetate transferase, SGPT: serum glutamate pyruvate transferase, and ALP: alkaline phosphatase.

The enzyme activities associated with liver functions are good biomarkers for the evaluation of hepatoprotective activity of medicinal plants. SGOT and SGPT are enzymes present in the hepatocytes, and their leakage into the blood stream is observed during cell damage. The level of SGOT is known to increase during cardiac or skeletal muscle damage. ALP is an enzyme which is present in the biliary duct lining of liver. The estimation of total bilirubin depicts the depth of jaundice and also indicates the severity of liver damage. The decrease in the total protein level is an indicator of liver damage caused by insignificant protein synthesis.

In the normal control group, the values of SGOT, SGPT, ALP, total and direct bilirubin, and protein content were found to be 59.83 ± 6.25, 52.83 ± 5.71, 101.5 ± 7.92 IU/L, 0.703 ± 0.05, 0.163 ± 0.02 mg/dL, and 8.47 ± 0.05 g/dL, respectively. However, the CCl₄ intoxicated control group displayed a significant increase in these values, like SGOT (145.83 ± 10.91 IU/L), SGPT (112.17 ± 7.47 IU/L), ALP (324.17 ± 8.70 IU/L), total bilirubin (1.42 ± 0.11 mg/dL), and direct bilirubin (0.34 ± 0.06 mg/dL), and a significant decrease in the total protein content (6.28 ± 0.08 g/dL). CPFA at a concentration of 400 mg/kg has shown a significant decrease in the serum enzymes like SGOT 59.83 ± 1.70 IU/L, SGPT 58.67 ± 2.63 IU/L, ALP 120.33 ± 2.63 IU/L, total bilirubin 0.638 ± 0.03 mg/dL, and direct bilirubin 0.14 ± 0.01 mg/dL and a significant increase in the total protein content 8.19 ± 0.02 g/dL, as compared to the toxic control group. A liver histopathological study also supported the protective effect of the extracts compared to the toxic control group. The images are represented in Figure 2.

Figure 2.

Photographs of hematoxylin/eosin-stained liver sections. (A) Normal control group rat representing normal liver architecture, (B) CCl₄ intoxicated rat liver showing cell necrosis around central vein, loss of cell boundaries, and ballooning degeneration, (C) liver section of the standard group showing less cell necrosis and less central vein crowding, (D) CPFA (200 mg/kg), and (E) CPFA (400 mg/kg) received group showed a moderate degree of liver damage and cell inflammation and reduced cell crowding.

Column chromatographic analysis of the extract was carried out with the universal solvent system that resulted in 13 different fractions. Phytochemical screening of these fractions revealed the presence of flavonoids, phenolics, and tannins. The results are depicted in Table 7.

Table 7. Phytochemical Screening for Phenolics and Flavonoids of Isolated Fractions of the Alcoholic Extract of C. paniculatum^a.

fractions	shinoda test	ferric chloride	lead acetate test	alkaline reagent test
F1	-	-	-	-
F 2	-	-	-	-
F 3	-	-	-	-
F 4	-	+	+	-
F 5	+++	++	+++	+++
F 6	-	+	++	+
F 7	++	+	+	+
F 8	++	++	+	+
F 9	+++	+++	+++	+++
F 10	++	+	++	+
F 11	+++	++	++	+
F 12	++	+++	+++	+
F 13	++	++	++	+

^a+++: highly present, ++: moderately present, +: low, -: absent, and F represents fractions.

Total phenolic and flavonoid contents were estimated for all the above fractions. Out of 13 fractions, fractions 5 and 9 showed the highest amount of flavonoids and phenolics. The total phenolic content of fraction 5 and 9 was 284.91 ± 6.03 and 378.31 ± 3.15 mg GAE/g, respectively, and their flavonoid content was evaluated as 333.82 ± 1.39 and 380.33 ± 1.55 mg QE/g, respectively (Table 8).

Table 8. Total Phenolic and Flavonoid Contents of Fractions Obtained from the Alcoholic Extract of C. paniculatum.

column fraction	total phenolic content (mg GAE/g)	flavonoid content (mg QE/g)
F 5	284.91 ± 6.03	333.82 ± 1.39
F 6	139.66 ± 1.93	198.39 ± 2.56
F 7	109.93 ± 3.48	115.02 ± 1.62
F 8	99.083 ± 4.49	99.74 ± 1.25
F 9	378.31±3.15	380.33±1.55
F 10	97.38 ± 2.18	116.70 ± 1.77
F 11	138.78 ± 4.85	174.50 ± 1.604
F 12	146.40 ± 0.50	178.56 ± 1.72
F 13	147.99 ± 1.06	192.52 ± 1.61

Their hepatoprotective evaluation was carried out by an in vitro model using goat liver slice culture for fractions 5 and 9 (highlighted in Table 8) that was estimated to have higher contents of phenolics and flavonoids. It was revealed that fraction 9 has much better activity, as compared to the toxic control group. [Table 9, Figure S5 (Supporting Information)].

Table 9. In Vitro Studies of Fractions^a.

groups	SGOT (IU/L)	SGPT (IU/L)	ALP (IU/L)	ACP (IU/L)
vehicle control	39.33 ± 3.512**	56.00 ± 2.646**	95.67 ± 2.082**	10.52 ± 1.572**
toxic control	191.00 ± 5.568	248.67 ± 2.082	309.00 ± 6.557	45.33 ± 1.528
standard (silymarin 50 mM)	42.67 ± 4.042**	62.00 ± 1.000**	106.67 ± 2.517**	12.33 ± 2.517**
CPLE extract (100 μg/mL)	150.00 ± 2.000**	210.00 ± 4.583**	266.00 ± 6.557**	41.67 ± 1.528**
CPLE extract (200 μg/mL)	128.33 ± 1.526**	180.00 ± 2.000**	221.33 ± 3.215**	36.33 ± 1.528**
CPLE extract (400 μg/mL)	101.33 ± 2.517**	157.33 ± 4.510**	181.67 ± 6.658**	33.00 ± 4.000**
fraction 5 (25 μg/mL)	132.00 ± 2.000**	156.33 ± 4.042**	172.67 ± 2.516**	40.67 ± 2.082**
fraction 5 (50 μg/mL)	124.00 ± 3.606**	142.67 ± 3.055**	161.33 ± 3.215**	31.67 ± 0.577**
fraction 5 (100 μg/mL)	96.33 ± 6.110**	132.33 ± 5.860**	145.67 ± 4.042**	30.00 ± 5.000**
fraction 9 (25 μg/mL)	95.67 ± 3.055**	124.00 ± 2.646**	147.00 ± 6.245**	27.33 ± 1.528**
fraction 9 (50 μg/mL)	70.33 ± 1.528**	110.67 ± 2.082**	132.33 ± 2.517**	26.00 ± 2.000**
fraction 9 (100 μg/mL)	56.67 ± 4.726**	83.0 ± 4.000**	118.00 ± 2.646**	20.00 ± 1.000**

^aThe values are represented as mean ± S.D (n = 3), **P < 0.05, as compared with the toxic control group.

GC–MS analyses of the fraction 5 and 9 were carried out to identify the compounds that are responsible for the exhibited activity. The spectrum of fraction 9 is shown in Figure 3 and fraction 5 in Figure S6 (Supporting Information). From the library search, it was revealed that the peak corresponding to 105.6 could be due to glyceric acid, peak of 169.2 could be due to gallic acid, peak of 207.35 could be due to pilocarpine, and peak of 281.39 could be due to pangamic acid.

Figure 3.

GC–MS chromatogram of fraction 9.

Further HPTLC analysis was carried out for the alcoholic extract of C. paniculatum flower. The active fractions 5 and 9, as separated by column chromatography along with standard quercetin were considered for evaluation. The solvent mixture chloroform/ethyl acetate/formic acid in the ratio 6:4:1 was used as the mobile phase. Silica gel 60 F 254 HPTLC plates were selected for analysis, and the plates were observed under white light and 254 and 366 nm. The presence of quercetin was confirmed and quantified. The images are depicted in Figure 4, and the respective chromatograms of the crude extract, the fractions under investigation along with the standard quercetin, are shown in Figure S7 (Supporting Information).

Figure 4.

HPTLC plates observed under (a) normal light and (b) 254 and (c) 366 nm.

As quantified from HPLC chromatogram under 254 nm, the amount of quercetin present in the alcoholic extract, fraction 5, and fraction 9 was 38.0, 34.6, and 49.6%, respectively.

Discussion

The liver, the major metabolic organ of our body, may have toxicity due to various drugs like alcohol, anabolic steroids, nonsteroidal anti-inflammatory drug, and so on.¹⁵ Oxidative stress can be attributed to the major reason behind this toxicity. Oxidative stress occurs due to the imbalance between free radicals generated and antioxidants present in the body.¹⁶ Many medicinal plants display significant implications toward traditional medicine for the treatment of hepatic diseases including hepatic disorders. Further these plants are being evaluated for in vivo pharmacological activities to identify potent candidates. The generation of free radicals as products of many biomolecular reactions plays major roles in the emergence of cancer and other health disturbances. It is also known that CCl₄ can induce the levels of various enzymes like ALT, AST, ALP, and γ-GT in animal models. CCl₄ is also known to cause acute hepatocyte injuries that further result in the leakage of various hepatocyte enzymes. Many hepatoprotective agents from different natural sources are reported to possess the ability to protect against such injuries by restoring the levels of the above enzymes along with retaining the levels of triglycerides, cholesterol, low-density lipoprotein, and high-density lipoprotein in the serum to normalcy. Earlier reports have also shown the potential of polyphenolic compounds to play key roles in establishing such protective activity.¹⁷ Several mechanisms were reported with animal models for the above observed activity with hepatoprotective agents that have strong ability to trap various metal ions like zinc, calcium, and iron.¹⁸ Herein, we have investigated the hepatoprotective effects of C. paniculatum plant bioactive compounds against CCl₄-induced hepatotoxicity in rats.

Internal antioxidant deficiency can be overcome by supplementing with external sources. Since polyphenolics are known to have high antioxidant potentials, their natural sources from plants have gained immense significance for them to be explored for extracting and isolating these polyphenolic antioxidant constituents.¹⁹ Among these antioxidants, flavonoids have a high health promoting role through their antioxidant mechanism. Due to their high free-radical scavenging property, the flavonoids display significant roles toward managing various diseases and disorders. Among the flavonoids, quercetin, rutin, apigenin, catechin, and so on are also reported for their anti-hepatotoxic properties.²⁰ Enzymatic and non-enzymatic components of the oxidative stress can be resolved through the plant cell defense system. A non-enzymatic system controls the cellular responses against the free radicals, whereas enzymatic responses directly scavenge the free radicals and hence control the antioxidant defense system.²¹ The qualitative chemical evaluation results obtained from our studies support the presence of various classes of chemical compounds like carbohydrates, proteins, flavonoids, tannins, phenolics, and steroids. The alcoholic extract of CPF exhibited high phenolics and flavonoids that influence the apparent antioxidant activity compared to other extracts. Herein, the antioxidant activity is evaluated by various methods including DPPH, nitric oxide radical scavenging, ABTS, and reducing power assay. The IC₅₀ value for the alcoholic extract was represented as 62.28 ± 0.51, 2362.71 ± 9.39 51, and 193.09 ± 5.84 μg/mL, respectively, for DPPH, ABTS, and nitric oxide radical scavenging methods. The alcoholic extracts were further subjected to hepatoprotective activity evaluation using CCl₄-induced hepatotoxic models. The evaluation was carried out by determining SGOT, SGPT, ALP, direct bilirubin, total bilirubin, and total protein content and also through histopathology of liver. The results obtained revealed a marked decrease in liver enzyme levels along with an increase in the total protein content when compared to toxic control groups that were similar to standard groups administered with silymarin. The total bilirubin in the serum serves as a hepatic functional marker which is associated with hepatic disorder along with acute disruption of hepatocellular architecture and function. Moreover, the toxicant-induced liver injuries also result in increased levels of bilirubin. The above findings are further supported by the results of histopathology analysis. In the toxic control group, the liver histopathological characters were showing disarranged hepatic cellular architecture with cell necrosis, fatty degeneration, and central vein crowding. This was almost reframed to the normal liver architecture by 200 mg/kg and 400 mg/kg body weight alcoholic extract treatment. In order to identify the unique constituents of the extract, the fractions displaying significant activities were separated by column chromatography using a universal solvent system (hexane, ethyl acetate, and methanol), and the fractions were tested for the presence of phenolics and flavonoid contents by both qualitative and quantitative techniques. Fractions 5 and 9 which showed better phenolic and flavonoid contents were then considered for in vitro hepatoprotective analyses using goat liver slice culture, and the activity was compared with the alcoholic extract of the plant. Of the two fractions, fraction 9 showed better activity. GC–MS analyses followed by NIST library search revealed that the constituents responsible for the above activity may be glyceric acid, gallic acid, pilocarpine, or pangamic acid, as shown in Figure 3. However, fraction 5 revealed the presence of quinovic acid as the active ingredient, and the GC–MS spectra of the same is given in Figure S6 (Supporting Information). Further HPTLC analysis revealed the presence of 34.6 and 49.6% quercetin in the active fractions 5 and 9, respectively, which was not revealed through GC–MS.

Conclusions

The implication on the pharmacological properties from the flower extract of C. paniculatum, family Verbenaceae, is reported for the first time in the literature. Various antioxidant activities carried out following various assays including DPPH, NO radical scavenging, ABTS activity, and reducing ability with the alcoholic extract showing significant activity compared to other extracts. The investigations by various enzymatic studies and histopathological sections with the alcoholic extract revealed excellent hepatoprotective activity. The best two fractions of the extract, as determined by column chromatographic analysis when subjected to in vitro hepatoprotective investigations, revealed fraction 9 to possess efficient hepatoprotective activity against carbon tetrachloride-induced liver toxicity in goat liver slice culture. Further analyses revealed that the hepatoprotective activities of C. paniculatum flower may be attributed to the presence of the following compounds that were isolated from fraction 9—(i) glyceric acid, (ii) gallic acid, (iii) pilocarpine, (iv) pangamic acid, and (v) quercetin either individually or in combination of the above.

Experimental Section

Chemicals and Reagents

The chemicals and reagents used for the experiments carried out for the present investigation were purchased from Spectrochem, SRL, Acros-Organics, RANKEM, and Fisher Scientific and used as received without further purification. ABTS, DPPH, and Folin-Ciocalteu’s reagents were obtained from Sigma-Aldrich.

Plant Collection

C. paniculatum flowers were collected from Malappuram, Kerala during August to October. The plant was identified and authenticated by Kottakkal Ayurveda research center, Kerala. The voucher specimen was deposited in the department for further reference.

Preparation of Plant Extracts

The flowers weighing ∼300 g were collected and dried under shade. Successively, solvent extraction was carried out using petroleum ether, chloroform, ethyl acetate, alcohol, and water as solvent medium following the hot continuous percolation method using a conventional Soxhlet apparatus. The extracts were concentrated, and the percentage yield was calculated.

Preliminary Phytochemical Screening

Preliminary qualitative analysis was carried out for detecting the presence of various classes of compounds like alkaloids, phenolics, flavonoids, saponins, glycosides, and steroids. Specific color change or any precipitate formation was considered as a positive response.^22,23

Determination of Total Phenolic and Flavonoid Contents

The Folin–Ciocalteau method was used for the quantitative determination of phenolics in each extract. 1 mL of the extract was mixed with 1 mL of Folin-Ciocalteu’s reagent. Then, 3 mL of sodium carbonate solution was added. The whole mixture was incubated for 2 h, and absorbance was measured at 725 nm.²⁴

The total flavonoid content was estimated using the aluminium chloride colorimetric method. 0.5 mL of the extract was mixed with 2 mL of water and 0.15 mL of NaNO₂ followed by the addition of 0.15 mL of 6% AlCl₃ solution. After 6 min, 2 mL of 4% sodium hydroxide solution was added, and volume was maintained to 5 mL using distilled water. Absorbance was measured at 510 nm.²⁵

In Vitro Assays

DPPH Method

The most effective and popular method of antioxidant evaluation is the DPPH radical scavenging method. Better activity is represented by the discoloration of DPPH solution. From stock solution of extracts, different concentrations ranging from 12.5 to 200 μg/mL were prepared. To 100 μL of the extract, 3.0 mL of DPPH solution was added and incubated at room temperature. After 20 min of incubation, absorbance was measured at 515 nm against methanol as blank. Absorbance of standard (ascorbic acid) was also measured. Percentage inhibition was measured by the following equation.²⁶

ABTS Method

In this method, the ABTS radical was generated by the oxidation of ABTS with potassium persulfate. For preparing ABTS solution, 7 mM aqueous ABTS solution and 2.45 mM potassium per sulfate were mixed together. The solution was incubated in the dark at 29 °C for 14 h. The working solution was prepared by diluting the previously prepared solution with phosphate buffer (pH 7.4). 50 μL of the extract was mixed with 3 mL of the freshly prepared solution and was allowed to stand for 20 min. After the incubation time, absorbance was measured at 734 nm. The percentage inhibition was measured using the above equation.²⁷

Nitric Oxide Radical Inhibitory Assay

Different concentrations of plant extracts were mixed with 1.5 mL of 5 mM sodium nitroprusside in phosphate buffered saline (pH 7.4). The solution was mixed together and incubated at 25 °C for 3 h. After the incubation time, 1.5 mL of the Griess reagent (2% phosphoric acid, 1% sulfanilamide, and 0.1% N-1-naphthyl ethylene diamine dihydrochloride) was added to the reaction mixture. Absorbance of the reaction mixture was measured at 546 nm with reference to standard gallic acid. The percentage inhibition was also measured.^28,29

Reducing Power Assay

In this method, better antioxidant activity was represented by an increase in the absorbance of the reaction mixture. Potassium ferricyanide, ferric chloride, and trichloroacetic acid produce a colored complex with the antioxidant compounds, and its absorbance can be measured at 700 nm. 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide were added to 10 mg/mL samples. The resulting mixture was incubated at 50 °C for 20 min. After the incubation period, 2.5 mL of trichloroacetic acid was added to the mixture, and the upper layer of the resulting mixture was collected by the 10 min centrifugation process. To 5 mL of above liquid, 5 mL of distilled water and 1 mL of ferric chloride (0.1% w/v) were added, and absorbance was measured at 700 nm.^30,31

Statistical Analysis

All the procedures were carried out in triplicates, and the results were expressed as the mean ± standard deviation. The statistical analyses were carried out using SPSS software version 20.

In Vivo Animal Experiments

Selection of Animals

Female Wistar Albino rats weighing 100–130 g were used for the hepatoprotective analysis of the alcoholic extract. Standard laboratory conditions were followed for animal maintenance, and they were fed with standard food and water ad libitum. The experimental protocol was approved by the Al Shifa College of Pharmacy Institutional Animal Ethical Committee (IAEC) (regd. no. 1195/Re/S/08/CPCSEA).

Acute Toxicity Study

C. paniculatum flower alcoholic extract’s acute toxicity was conducted on albino rats, according to the OECD guidelines no. 425. After 12 h of fasting, the extract (2000 mg/kg) was administered orally. The animals were observed for 14 days to check mortality, any behavioral changes, any discomforts, and so on.³²

CCl₄-Induced Hepatoprotective Study

Five groups of six animals were selected, and the experiment was conducted for 14 days.

Doses: the animals of the vehicle group (group I) received CMC (5 ml/kg b.w.) orally, and CCl₄ in liquid paraffin was administered to animals of group II–V via subcutaneous route for 14 days. Standard dose (silymarin, 50 mg/kg b.w.) was administered to group III, and group IV animals received CPFA1 (200 mg/kg b.w.) by oral route. Group V animals received the CPFA 2 (400 mg/kg b.w.) extract. After 14 days of treatment, all the animals were sacrificed by cervical decapitation, and blood samples were collected by cardiac puncture. Serum was collected by centrifugation at 3000 rpm and examined for enzyme analysis such as SGOT, SGPT, ALP, total and direct bilirubin, and total protein content. Data were analyzed by one-way ANOVA followed by Dunnett’s test.³³⁻³⁵

Statistical Analysis

The results of the in vivo experiments conducted were expressed as mean ± S.E.M. SPSS software version 20 was used for statistical analysis.

Histopathology

The livers were excised and washed with normal saline. Liver fragments were fixed in 10% buffered formalin followed by paraffin embedding. Liver sections of 0.5 μm thickness were taken and stained with the hematoxylin–eosin dye. The sections were mounted and microscopically observed for studying histological changes if any.³⁶

Isolation of Compound Using Column Chromatography

Around 1 g of the alcoholic extract of the plant was loaded into the column, and gradient elution was carried out using hexane, ethyl acetate, and methanol as the mobile phase.^37,38 The solvent was passed through the column at 1 mL per minute under gravity to fractionate the sample extract. Each fraction was collected in a test tube and was numbered subsequently. The fractions obtained were subjected to the qualitative chemical test for identification of tannins, phenolics, and flavonoids.^22,23,39 The quantitative estimation for phenolics and flavonoid was carried out,^24,25 and the best two fractions were selected for an in vitro hepatoprotective activity study using goat liver slice culture.

In Vitro Hepatoprotective Study

The fresh goat livers were obtained from the local market. The liver was removed and transferred to presterilized Krebs Ringer Herpes (KRH) medium. The liver was cut into thin slices ranging from 4 to 6 mg using a sharp blade and was used for the study. Each set weighing 100 mg contains 20–25 slices. Tissues were washed with 10 mL of KRH medium in every 10 min over a period of 1 h. The slices were preincubated at 37 °C for 60 min in cotton-plugged beakers containing 10 mL of KOH. The liver slices were further divided into individual culture for respective treatment. All the cultures were incubated at constant temperature in a water bath at 37 °C for 2 h. The cells were isolated from the culture medium of each set by centrifuging at 3000 rpm for 10 min at 4 °C, and the corresponding supernatants were assayed for the presence of leaked biochemical markers such as alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and acid phosphatase (ACP).⁴⁰⁻⁴³

Experimental Setup for Evaluation of Hepatoprotective Activity

Group 1: normal control, Group 2: toxic control—CCl₄ (15.5 mM), Group 3: standard (silymarin-50 mM), Group 4–6:C. paniculatum alcoholic extract at the concentration range of 25–100 μg/mL, Group 7–9: column fraction 5 at the concentration range of 2.5–10 μg/mL, and Group 10–12: column fraction 9 at the concentration range of 2.5–10 μg/mL.

GC–MS Analyses

Mass spectra were recorded on a Schimadzu GCMS-QP2020 Gas chromatograph mass spectrometer. The constituents present in various fractions of the extracts were predicted using NIST/EPA/NIH mass spectral library—2017. The equipment has a DB 35-MS capillary standard non-polar poly (dimethylsiloxane) column with dimensions of 30 mm × 0.25 mm ID × 0.25 μm film. Helium was used as the carrier gas with a flow rate of 1.0 mL/min. The injector was operated at 280 °C, and the oven temperature was programmed as follows: 70–280 °C, gradually increased by 10 °C per/min.

Glossary

Abbreviations

CPFP

Clerodendrum paniculatum flower petroleum ether extract

CPFC

Clerodendrum paniculatum flower chloroform extract

CPFE

Clerodendrum paniculatum flower ethyl acetate extract

CPFA

Clerodendrum paniculatum flower alcohol extract

CPFW

Clerodendrum paniculatum flower water extract

DPPH

2,2-diphenyl-2-picryl hydrazyl

ABTS

2,2-azino-bis (3-ethyl benzothiazoline-6-sufonic acid)

NO

nitric oxide

GAE

gallic acid equivalent

QE

quercetin equivalent

SGOT

serum glutamate oxaloacetate transaminase

SGPT

serum glutamate pyruvate transaminase

ALP

alkaline phospahtase

γ-GT

gamma glutamyl transferase

CMC

carboxy methyl cellulose

b.w.

body weight

s.c

subcutaneous

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c03722.

• Antioxidant activity and GC–MS spectrum of fraction 5 (PDF)

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

The authors declare no competing financial interest.