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Efficacy evaluation of a Chinese bitter tea (Ilex latifolia Thunb.) via analyses of its main components Ting Hu, Xiao-Wei He, Jian-Guo Jiang* and Xi-Lin Xu* In order to evaluate the health effects of Ilex latifolia Thunb., a Chinese bitter tea widely consumed as a health beverage, flavonoids, saponins, polysaccharides and alkaloids were extracted from its leaves and their in vitro antioxidant activity, anticomplement system activity, antiproliferative effects against human cervical carcinoma Hela cells, and anti-inflammatory effects against mouse macrophage RAW 264.7 cells were analyzed. Results showed that the polysaccharides exhibited a considerable inhibition of the complement system, the hemolysis inhibition rate reached 98% at a concentration of 0.8 mg mL1, which was clearly higher than that of the positive control (heparin sodium). The total flavonoids displayed significant DPPH scavenging activity and an inhibition effect on the generation of NO in LPS-
Received 18th November 2013 Accepted 25th January 2014
induced RAW 264.7 macrophages. In addition, the total saponins showed a better antiproliferative effect against Hela cells, and the total alkaloids exhibited a high reducing power. It is obvious that I. latifolia has
DOI: 10.1039/c3fo60603a
a variety of nutritional and health functions which are attributed to its different components. The analysis
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method presented in this research can suggest lessons for analysis of other plant foods.
1. Introduction Ilex latifolia Thunb., a traditional Chinese bitter tea, is widely consumed in East Asia.1 It is called kudingcha in Chinese. I. latifolia has been used as a tea beverage in China for more than 2000 years, and in traditional Chinese medicine, it is also prescribed for the treatment of obesity, hypertension, cardiovascular disease, hyperlipidemia and other diseases, the treatment of which is attributed to a variety of active phytochemical constituents in the plants.2 Over the last several years, kudingcha has been considered as a dietetic beverage and is gaining popularity with names like “beauty-slimming tea”, “longevity tea”, and “depressurization tea”. It tastes bitter followed by sweet. Studies have shown that drinking tea from I. latifolia for 2 months is sufficient to reduce hypertension nearly as well as or better than common hypertension medications such as nifedipine. More importantly, no adverse reaction was observed.1 Plants are generally believed to be rich in a wide variety of secondary metabolites such as alkaloids, avonoids, polysaccharides and saponins, which are the natural active ingredients and their pharmacological activity is different in general. Flavonoids from tea are powerful antioxidant compounds that can help the human body to reduce the risks of stroke, heart failure, diabetes and cancer.3 Saponins have been used in the food and cosmetic industries because of their foaming and College of Food and Bioengineering, South China University of Technology, Guangzhou, 510640, China. E-mail: [email protected]; [email protected]; Fax: +86-20-87113843; +86-20-87113842; Tel: +86-20-87113849; +86-20-87113848
876 | Food Funct., 2014, 5, 876–881
emulsifying properties. The Quillaja bark saponin is an approved ingredient for use in food and beverages as a avoring agent.4 Triterpenoid saponins from the leaves of I. latifolia can inhibit the LDL-induced formation of foam cells and reduce intracellular total cholesterol and triglyceride contents.5 Polysaccharides from botanicals have shown a variety of bioactivities. T. matsutake polysaccharides have displayed multiple functions including immunoregulation, antitumour, antioxidative, anti-mutagenic, and hematopoietic activities.6 Alkaloids also have a wide range of physiological and pharmacological activity, although their content is oen relatively low in plants. In order to analyze the various potential health functions of I. latifolia and their relationship with its main constituents, in the present study, four chemical constituents (avonoids, saponins, polysaccharides and alkaloids) were obtained from the leaves of I. latifolia in order to compare their potential activities of antioxidation, anticomplement system, antiproliferation against human cervical carcinoma Hela cells, and anti-inammation against RAW 264.7 macrophages. It is hoped that the investigation method will be benecial for the evaluations of other natural plant foods.
2.
Results and discussion
2.1
Complement hemolytic activity assay
The human complement system plays an important role in the host defense system against foreign invasive organisms and in external wounds. Its effects are normally benecial to the host,
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I. latifolia has been recognized as a good source of phytochemicals, and four extracts were assessed for their antioxidant effects using the DPPH free radical scavenging assay and the FRAP assay. Some model assays, like the hydroxyl radical and superoxide anion, have certain limitations and drawbacks from a mechanistic point of view. The peroxyl radicals are the most
common free radicals in vivo; they are highly reactive and unstable. The free radicals used in the DPPH assay are stable organic nitrogen radicals and bear no similarity to peroxyl radicals. Fig. 2 shows the DPPH free radical scavenging ability of each sample. All chemical constituents extracted from I. latifolia exhibited a certain amount of scavenging activity. The scavenging rate of total avonoids rose sharply along with an increase in concentration and approached the positive control (Vc) at the highest concentration (0.8 mg mL1). The total polysaccharides and total alkaloids showed a better scavenging ability just lower than the total avonoids at high concentrations (0.4–0.8 mg mL1), while they both exceeded the total avonoids at low concentrations (0.05–0.2 mg mL1). The DPPH scavenging ability of total saponins was lower than the other three extracts, but it exhibited some activity. In recent years, avonoids with antioxidant effects and the effect of scavengingfree radicals have been widely appreciated by the pharmaceutical industry. Flavonoids are responsible for the antioxidant activities of numerous botanical extracts. They can protect the human body from free radicals and inhibit the progression of many chronic diseases.9 The results we obtained seemed to agree with those of a previous study, which showed that the total avonoids from Cichorium glandulosum seeds exhibited an excellent scavenging activity.10 The results of a FRAP assay are shown in Fig. 3. The antioxidant capacity increased with increasing concentration both for the constituents and the Vc control. The antioxidant activity of the Vc control sharply responded to concentration, whereas that of the constituents changed gently. Comparatively speaking, among the four chemical constituents extracted from I. latifolia, the total alkaloids showed the highest antioxidant capacity, followed by total polysaccharides and total avonoids. Although the action of all of the extracts was lower than the positive control (Vc), the FRAP value of total alkaloids was close to Vc at a concentration of 0.8 mg mL1. Total avonoids and total polysaccharides showed reducing power to a certain extent, while total saponins exhibited a lower reducing capacity. In the present study, many phytochemical constituents were reported to display a better reducing capacity of antioxidants
The inhibitory effects of flavonoids, saponins, polysaccharides and alkaloids extracted from I. latifolia on the classical pathway of the complement system.
Fig. 2 The DPPH free radical scavenging activity of flavonoids, saponins, polysaccharides and alkaloids. Results are mean SD. *P < 0.05, **P < 0.01, statistically significant in comparison with the control (Vc).
but it can also cause adverse effects depending on the site, extent, and duration of complement activation. Activation of the system may lead to a pathologic reaction in a variety of inammatory and degenerative diseases. Different chemical constituents extracted from I. latifolia, including total avonoids, total saponins, total polysaccharides and total alkaloids, were tested for their anticomplement activity via the classical pathway of the complement system, the results of which are presented in Fig. 1. Among them, total polysaccharides exhibited a very strong inhibitory effect on the complement system with the hemolysis inhibition rate reaching 98% at a concentration of 0.8 mg mL1, which was considerably higher than that of the positive control (heparin sodium). Conversely, total saponins and total alkaloids did not exhibit hemolytic activity. Moreover, the total avonoids showed to a certain extent anticomplement activity. It was previously reported that a sulfated derivative prepared from homogalacturonan, which is a polysaccharide found in the fruits of Capparis spinosa L., had signicant anticomplement activity.7 In addition, the crude polysaccharides isolated from the root of Bupleurum smithii var. parvifolium had anticomplement activity and immunomodulatory functions.8 Anticomplement activity reects the immune system. This observation that the total polysaccharides from I. latifolia played a crucial role in the anticomplement activity, was consistent with the concept that polysaccharides isolated from plants or edible fungi generally have immune activity. These results indicated that I. latifolia polysaccharides could be considered as a promising candidate for an anticomplement medicine, since it is efficient at inhibiting the complement system at a certain concentration. 2.2
Antioxidant assays
Fig. 1
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Fig. 3 The ferric reducing/antioxidant power activity of flavonoids, saponins, polysaccharides and alkaloids. Results are mean SD. *P < 0.05, **P < 0.01, statistically significant in comparison with the control (Vc).
while there were no such ndings for the total alkaloids. Most of the known functions of alkaloids are related to protection. Alkaloid-related substances such as serotonin, dopamine and histamine are important neurotransmitters in animals. I. latifolia is used as a tea in our daily life, like green tea, which contains abundant tea polyphenols, maybe it is the primary cause that it has a strong antioxidant capacity. The results we obtained suggest that I. latifolia has the potential active phytochemicals of antioxidation, for application in functional foods and cosmetics.
2.3
Antitumor activity
Oxidative stress is one of the main causes of cancer-related death, and chemoprevention is dened as the use of natural or synthetic antioxidants such as triterpenoid saponines, avonoids and sterols to prevent the formation of cancer or the development of cancer.9 In this investigation, the chemical constituents extracted from I. latifolia were evaluated for their antiproliferative activities. Fig. 4 shows the cell proliferation inhibition rate of each sample. Different constituents showed different inhibition effects on the proliferation of cervical
Fig. 4 The cell proliferation inhibition rate of flavonoids, saponins,
polysaccharides and alkaloids. Results are mean SD. *P < 0.05, **P < 0.01, statistically significant in comparison with the control (5-FU).
878 | Food Funct., 2014, 5, 876–881
Paper
cancer Hela cells. Among the four extracts, total avonoids and total saponins both exhibited the strongest cytotoxicity. In particular, total saponins could signicantly inhibit the Hela cells at concentrations ranging from 50 to 200 mg mL1. The antitumor activity occurred in a concentration-dependent manner. Although the inhibition rates of all of the extracts were lower than that of the positive control (5-uorouracil), the inhibition rate of total saponins reached 50%, and total avonoids reached 78%, which was near to that of 5-uorouracil, at a concentration of 200 mg mL1. Nevertheless, total polysaccharides and total alkaloids showed a general effect. These data suggest that I. latifolia has an inuence on tumor cell viability and targeted Hela cell lines, indicating the presence of powerful cytotoxic compounds in the saponins and avonoids. Kicha et al.11 showed that two asterosaponins isolated from the Vietnamese starsh Archaster typicus had a high anticancer activity against human cervix carcinoma Hela cell lines. Bhagat et al.12 obtained similar results from the Eucalyptus citriodora leaves, showing that the extracts displayed a very strong antiproliferative effect against Hela cell lines, which may be due to the avonoids and saponins that are present in the plant. Altogether, these results suggest that I. latifolia had appreciable antitumor activity and might be considered as a potential source of anticancer compounds mainly from the saponins and avonoids.
2.4 Effect of the extracts on NO production in RAW 264.7 macrophages Current studies have demonstrated the participation of reactive oxygen species in models of inammation. I. latifolia was investigated as a potential inhibitor of NO production in inammatory reactions. LPS-induced stimulation of RAW 264.7 macrophages led to overproduction of NO, which could be detected and quantied. The results presented in Fig. 5 show that all of the extracts signicantly inhibited NO release and decreased in the order of total avonoids (97%), total saponins (85.25%), total polysaccharides (73.22%) and total alkaloids
Fig. 5 Inhibitory effect on NO production of flavonoids, saponins, polysaccharides and alkaloids extracted from I. latifolia in LPS-induced RAW 264.7 macrophages. Results are mean SD. *P < 0.05, **P < 0.01, statistically significant in comparison with the three others.
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(53.87%) at a concentration of 200 mg mL1. It was noted that total avonoids exhibited the most active anti-inammatory effect among all of the extracts. Even at the lowest concentration (12.5 mg mL1) it also exhibited a better inhibitory effect of above 50%. Lai et al.13 showed that a avonoid compound (formononetin) isolated from the root of Astragalus membranaceus could signicantly inhibit NO production in LPSinduced RAW 264.7 macrophages. In addition, bioactive compounds like saponins have been reported to be benecial in the treatment of chronic inammatory diseases associated with the overproduction of NO. In conclusion, the four main constituents, avonoids, saponins, polysaccharides and alkaloids extracted from I. latifolia exhibited different levels of antioxidant, anticomplement, antitumor and anti-inammatory activities. Total polysaccharides showed an obvious inhibitory effect on the complement system, even stronger than the positive control. The total avonoids displayed signicant DPPH scavenging activity and anti-inammatory effects on LPS-induced RAW 264.7 macrophages. The total saponins showed a better antiproliferative effect against human cervical carcinoma Hela cells, and the total alkaloids exhibited higher reducing power. The results provide useful information on the pharmacological activities of this traditional herbal tea. With the demand for healthy and nutritious plant foods, the nutraceutical market exploring the potential health benets of I. latifolia products is expanding. From a health point of view, kudingcha made from I. latifolia is a useful variety of herbal drinks, due to its various biological activities, suggesting its good potential for the development of dietary supplements. The mechanisms of anticomplement activity of polysaccharides, antioxidation of saponins, and antiproliferation of avonoids are worthy of further research. To the best of our knowledge, this is the rst study to comprehensively evaluate I. latifolia. The results reported here may give researchers insight and guidance in designing future in vivo studies on the health benets associated with the regular intake of I. latifolia.
3. 3.1
Materials and methods Materials
I. latifolia was collected from Wuzhi Mountain, Hainan, China on September 15th, 2012, and was authenticated by Professor Dingyong Wang of the Chemistry department, Guangdong College of Pharmacy, where voucher specimens were kept. Samples were air-dried under shade for one week and ground into a ne powder with a laboratory pulverizer. The dried materials were stored at room temperature in desiccators until use. All other reagents used in the experiments were of analytical grade.
For total avonoids and saponins, the powder (100 g) of I. latifolia was extracted with 80% ethanol for 1 h. The crude extract was evaporated under vacuum to dryness as a dark brown mass. The separation and purication of the two constituents was carried out by using AB-8 macroporous resin eluted with a gradient of ethanol and water (0 : 100, 20 : 80, 50 : 50, 70 : 30, 100 : 0, v/v). The total avonoids fraction (70 : 30 eluates, 6.2 g) and total saponins fraction (50 : 50 eluates, 4.8 g) were obtained according to previous literatures. For the total polysaccharides, the extraction process of polysaccharides (100 g powder) was performed as for the previous method of water extraction and alcohol precipitation.14 The protein was removed from the crude extract and the total polysaccharides sample was obtained (9.6 g). For the total alkaloids, the powder (100 g) of I. latifolia was extracted with an ethanol solution containing a low concentration of hydrochloric acid. The extracts were ltered and concentrated, followed by dissolving in 2% hydrochloric acid, the pH was adjusted to 2–3 and then the insoluble substance was removed. The supernate was extracted with chloroform twice, the chloroform phase was removed, the pH was adjusted to 9–10 using ammonia, and extracted with chloroform until there were no alkaloids in the chloroform phase. The extracts were combined and concentrated to obtain the total alkaloids (2.3 g). The extraction and separation procedure of total avonoids, total saponins, total polysaccharides and total alkaloids from I. latifolia is shown in Fig. 6. 3.3 Determination of the content of the four chemical constituents Modication of the method by Deng et al.15 was conducted to determine the content of the total avonoids at 510 nm. The calibration curve prepared using rutin standard solutions was obtained as Y ¼ 13.113X 0.0177 (R2 ¼ 0.9994), the linear range was 0.01–0.07 mg mL1. The content of the saponins was determined by UV/vis spectrophotometry with ginsenoside Rb1 as the standard examined at 545 nm.15 The regression equation of the standard curve was obtained as Y ¼ 17.925X 0.0498 (R2 ¼ 0.9988), the linear range was 0.05–0.3 mg mL1. The content of the total polysaccharides was conducted using the phenol– sulfuric acid method with D-glucose as the standard at 490 nm,14 which was prepared in sequential concentrations to make a standard curve for the polysaccharide calculation. The regression equation was obtained as Y ¼ 13.821X + 0.0433 (R2 ¼ 0.999), the linear range was 0.01–0.07 mg mL1. Berberine hydrochloride was used to determine the content of the alkaloids as the reference substance that was measured using a colorimetric assay of an acid dye.16 The regression equation of the standard curve was obtained as Y ¼ 7.4484X + 0.0009 (R2 ¼ 0.999), the linear range was 0.04–0.2 mg mL1.
3.2 Extraction of total avonoids, saponins, polysaccharides and alkaloids
3.4
For the purpose of meeting the requirements of the bioactivity assays the four kinds of constituents (avonoids, saponins, polysaccharides and alkaloids) were extracted from I. latifolia in the following ways.
The anticomplement assay through the classical pathway was conducted according to Seo's17 modied method, which was measured by the complement xation test based on complement consumption and the degree of red blood cell lysis by the
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Anticomplement activity
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Fig. 6
The extraction and separation procedure of total flavonoids, total saponins, total polysaccharides and total alkaloids from I. latifolia.
residual complement. The optical density of the supernatant was measured at 405 nm and heparin sodium was used as the positive control. 3.5
Antioxidant assays
3.5.1 Radical scavenging activity of DPPH. The scavenging activity of the DPPH free radical was estimated according to the method of Mu˜ niz-Marquez et al.18 with minor modications. The absorbance of the mixture was measured at 517 nm with a UV-visible spectrophotometer. A decrease of the absorbance indicated an increase of DPPH radical scavenging activity. Analysis of all of the samples was carried out in triplicate with ascorbic acid (Vc) as the positive control. The ability of the samples to scavenge the DPPH free radicals was calculated according to the equation: As Ac DPPH radical scavengingð%Þ ¼ 1 100% Ab Ac where As, Ac and Ab represent the absorbance of the sample, the control and the blank groups, respectively. 3.5.2 Ferric reducing/antioxidant power (FRAP) assay. The ferric reducing/antioxidant power of each sample was measured according to the method of Kapoor et al.19 An increase in antioxidant activity was indicated by increasing absorbance. The absorbance of the mixture solution was measured at 700 nm. Each sample was run in triplicate and averaged. 3.6
Paper
Antitumor activity
The human cervix carcinoma Hela cells were maintained and cultured in tissue culture asks containing DMEM medium
880 | Food Funct., 2014, 5, 876–881
supplemented with 10% fetal bovine serum, 1% penicillin (100 IU mL1) and streptomycin (100 mg mL1) at 37 C in a humidied incubator with 5% CO2. The effect of each sample on the proliferation of Hela cells was estimated using the MTT assay.20 5-Fluorouracil was used as the positive control. The cell proliferation inhibition rate was calculated using the following formula: Cell proliferation inhibitionð%Þ ¼ 3.7
1
ODsample ODcontrol
100%
Anti-inammatory assays
RAW 264.7 macrophages were cultured in DMEM supplemented with 10% fetal bovine serum, 1% penicillin (100 IU mL1) and streptomycin (100 mg mL1) in a 5% CO2 atmosphere at 37 C. The cells were activated with LPS as in the method described by Hsu et al.21 To assess NO production, the stable conversion product of NO was measured using the Griess reagent and the optical density was determined at 540 nm. The NO inhibition rate was calculated using the following formula: NO inhibitionð%Þ ¼ 3.8
ODLPS ODLPSþsample 100% ODLPS ODblank
Statistical analysis
All of the results are expressed as the means of the three experiments standard deviations (SD). The statistical signicance of the differences between the means was assessed by a one-way analyses of variance (ANOVA). P < 0.05 indicated the presence of a statistically signicant difference and P < 0.01 was considered highly signicant.
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Efficacy evaluation of a Chinese bitter tea (Ilex latifolia Thunb.) via analyses of its main components Ting Hu, Xiao-Wei He, Jian-Guo Jiang* and Xi-Lin Xu* In order to evaluate the health effects of Ilex latifolia Thunb., a Chinese bitter tea widely consumed as a health beverage, flavonoids, saponins, polysaccharides and alkaloids were extracted from its leaves and their in vitro antioxidant activity, anticomplement system activity, antiproliferative effects against human cervical carcinoma Hela cells, and anti-inflammatory effects against mouse macrophage RAW 264.7 cells were analyzed. Results showed that the polysaccharides exhibited a considerable inhibition of the complement system, the hemolysis inhibition rate reached 98% at a concentration of 0.8 mg mL1, which was clearly higher than that of the positive control (heparin sodium). The total flavonoids displayed significant DPPH scavenging activity and an inhibition effect on the generation of NO in LPS-
Received 18th November 2013 Accepted 25th January 2014
induced RAW 264.7 macrophages. In addition, the total saponins showed a better antiproliferative effect against Hela cells, and the total alkaloids exhibited a high reducing power. It is obvious that I. latifolia has
DOI: 10.1039/c3fo60603a
a variety of nutritional and health functions which are attributed to its different components. The analysis
www.rsc.org/foodfunction
method presented in this research can suggest lessons for analysis of other plant foods.
1. Introduction Ilex latifolia Thunb., a traditional Chinese bitter tea, is widely consumed in East Asia.1 It is called kudingcha in Chinese. I. latifolia has been used as a tea beverage in China for more than 2000 years, and in traditional Chinese medicine, it is also prescribed for the treatment of obesity, hypertension, cardiovascular disease, hyperlipidemia and other diseases, the treatment of which is attributed to a variety of active phytochemical constituents in the plants.2 Over the last several years, kudingcha has been considered as a dietetic beverage and is gaining popularity with names like “beauty-slimming tea”, “longevity tea”, and “depressurization tea”. It tastes bitter followed by sweet. Studies have shown that drinking tea from I. latifolia for 2 months is sufficient to reduce hypertension nearly as well as or better than common hypertension medications such as nifedipine. More importantly, no adverse reaction was observed.1 Plants are generally believed to be rich in a wide variety of secondary metabolites such as alkaloids, avonoids, polysaccharides and saponins, which are the natural active ingredients and their pharmacological activity is different in general. Flavonoids from tea are powerful antioxidant compounds that can help the human body to reduce the risks of stroke, heart failure, diabetes and cancer.3 Saponins have been used in the food and cosmetic industries because of their foaming and College of Food and Bioengineering, South China University of Technology, Guangzhou, 510640, China. E-mail: [email protected]; [email protected]; Fax: +86-20-87113843; +86-20-87113842; Tel: +86-20-87113849; +86-20-87113848
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emulsifying properties. The Quillaja bark saponin is an approved ingredient for use in food and beverages as a avoring agent.4 Triterpenoid saponins from the leaves of I. latifolia can inhibit the LDL-induced formation of foam cells and reduce intracellular total cholesterol and triglyceride contents.5 Polysaccharides from botanicals have shown a variety of bioactivities. T. matsutake polysaccharides have displayed multiple functions including immunoregulation, antitumour, antioxidative, anti-mutagenic, and hematopoietic activities.6 Alkaloids also have a wide range of physiological and pharmacological activity, although their content is oen relatively low in plants. In order to analyze the various potential health functions of I. latifolia and their relationship with its main constituents, in the present study, four chemical constituents (avonoids, saponins, polysaccharides and alkaloids) were obtained from the leaves of I. latifolia in order to compare their potential activities of antioxidation, anticomplement system, antiproliferation against human cervical carcinoma Hela cells, and anti-inammation against RAW 264.7 macrophages. It is hoped that the investigation method will be benecial for the evaluations of other natural plant foods.
2.
Results and discussion
2.1
Complement hemolytic activity assay
The human complement system plays an important role in the host defense system against foreign invasive organisms and in external wounds. Its effects are normally benecial to the host,
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I. latifolia has been recognized as a good source of phytochemicals, and four extracts were assessed for their antioxidant effects using the DPPH free radical scavenging assay and the FRAP assay. Some model assays, like the hydroxyl radical and superoxide anion, have certain limitations and drawbacks from a mechanistic point of view. The peroxyl radicals are the most
common free radicals in vivo; they are highly reactive and unstable. The free radicals used in the DPPH assay are stable organic nitrogen radicals and bear no similarity to peroxyl radicals. Fig. 2 shows the DPPH free radical scavenging ability of each sample. All chemical constituents extracted from I. latifolia exhibited a certain amount of scavenging activity. The scavenging rate of total avonoids rose sharply along with an increase in concentration and approached the positive control (Vc) at the highest concentration (0.8 mg mL1). The total polysaccharides and total alkaloids showed a better scavenging ability just lower than the total avonoids at high concentrations (0.4–0.8 mg mL1), while they both exceeded the total avonoids at low concentrations (0.05–0.2 mg mL1). The DPPH scavenging ability of total saponins was lower than the other three extracts, but it exhibited some activity. In recent years, avonoids with antioxidant effects and the effect of scavengingfree radicals have been widely appreciated by the pharmaceutical industry. Flavonoids are responsible for the antioxidant activities of numerous botanical extracts. They can protect the human body from free radicals and inhibit the progression of many chronic diseases.9 The results we obtained seemed to agree with those of a previous study, which showed that the total avonoids from Cichorium glandulosum seeds exhibited an excellent scavenging activity.10 The results of a FRAP assay are shown in Fig. 3. The antioxidant capacity increased with increasing concentration both for the constituents and the Vc control. The antioxidant activity of the Vc control sharply responded to concentration, whereas that of the constituents changed gently. Comparatively speaking, among the four chemical constituents extracted from I. latifolia, the total alkaloids showed the highest antioxidant capacity, followed by total polysaccharides and total avonoids. Although the action of all of the extracts was lower than the positive control (Vc), the FRAP value of total alkaloids was close to Vc at a concentration of 0.8 mg mL1. Total avonoids and total polysaccharides showed reducing power to a certain extent, while total saponins exhibited a lower reducing capacity. In the present study, many phytochemical constituents were reported to display a better reducing capacity of antioxidants
The inhibitory effects of flavonoids, saponins, polysaccharides and alkaloids extracted from I. latifolia on the classical pathway of the complement system.
Fig. 2 The DPPH free radical scavenging activity of flavonoids, saponins, polysaccharides and alkaloids. Results are mean SD. *P < 0.05, **P < 0.01, statistically significant in comparison with the control (Vc).
but it can also cause adverse effects depending on the site, extent, and duration of complement activation. Activation of the system may lead to a pathologic reaction in a variety of inammatory and degenerative diseases. Different chemical constituents extracted from I. latifolia, including total avonoids, total saponins, total polysaccharides and total alkaloids, were tested for their anticomplement activity via the classical pathway of the complement system, the results of which are presented in Fig. 1. Among them, total polysaccharides exhibited a very strong inhibitory effect on the complement system with the hemolysis inhibition rate reaching 98% at a concentration of 0.8 mg mL1, which was considerably higher than that of the positive control (heparin sodium). Conversely, total saponins and total alkaloids did not exhibit hemolytic activity. Moreover, the total avonoids showed to a certain extent anticomplement activity. It was previously reported that a sulfated derivative prepared from homogalacturonan, which is a polysaccharide found in the fruits of Capparis spinosa L., had signicant anticomplement activity.7 In addition, the crude polysaccharides isolated from the root of Bupleurum smithii var. parvifolium had anticomplement activity and immunomodulatory functions.8 Anticomplement activity reects the immune system. This observation that the total polysaccharides from I. latifolia played a crucial role in the anticomplement activity, was consistent with the concept that polysaccharides isolated from plants or edible fungi generally have immune activity. These results indicated that I. latifolia polysaccharides could be considered as a promising candidate for an anticomplement medicine, since it is efficient at inhibiting the complement system at a certain concentration. 2.2
Antioxidant assays
Fig. 1
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Fig. 3 The ferric reducing/antioxidant power activity of flavonoids, saponins, polysaccharides and alkaloids. Results are mean SD. *P < 0.05, **P < 0.01, statistically significant in comparison with the control (Vc).
while there were no such ndings for the total alkaloids. Most of the known functions of alkaloids are related to protection. Alkaloid-related substances such as serotonin, dopamine and histamine are important neurotransmitters in animals. I. latifolia is used as a tea in our daily life, like green tea, which contains abundant tea polyphenols, maybe it is the primary cause that it has a strong antioxidant capacity. The results we obtained suggest that I. latifolia has the potential active phytochemicals of antioxidation, for application in functional foods and cosmetics.
2.3
Antitumor activity
Oxidative stress is one of the main causes of cancer-related death, and chemoprevention is dened as the use of natural or synthetic antioxidants such as triterpenoid saponines, avonoids and sterols to prevent the formation of cancer or the development of cancer.9 In this investigation, the chemical constituents extracted from I. latifolia were evaluated for their antiproliferative activities. Fig. 4 shows the cell proliferation inhibition rate of each sample. Different constituents showed different inhibition effects on the proliferation of cervical
Fig. 4 The cell proliferation inhibition rate of flavonoids, saponins,
polysaccharides and alkaloids. Results are mean SD. *P < 0.05, **P < 0.01, statistically significant in comparison with the control (5-FU).
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Paper
cancer Hela cells. Among the four extracts, total avonoids and total saponins both exhibited the strongest cytotoxicity. In particular, total saponins could signicantly inhibit the Hela cells at concentrations ranging from 50 to 200 mg mL1. The antitumor activity occurred in a concentration-dependent manner. Although the inhibition rates of all of the extracts were lower than that of the positive control (5-uorouracil), the inhibition rate of total saponins reached 50%, and total avonoids reached 78%, which was near to that of 5-uorouracil, at a concentration of 200 mg mL1. Nevertheless, total polysaccharides and total alkaloids showed a general effect. These data suggest that I. latifolia has an inuence on tumor cell viability and targeted Hela cell lines, indicating the presence of powerful cytotoxic compounds in the saponins and avonoids. Kicha et al.11 showed that two asterosaponins isolated from the Vietnamese starsh Archaster typicus had a high anticancer activity against human cervix carcinoma Hela cell lines. Bhagat et al.12 obtained similar results from the Eucalyptus citriodora leaves, showing that the extracts displayed a very strong antiproliferative effect against Hela cell lines, which may be due to the avonoids and saponins that are present in the plant. Altogether, these results suggest that I. latifolia had appreciable antitumor activity and might be considered as a potential source of anticancer compounds mainly from the saponins and avonoids.
2.4 Effect of the extracts on NO production in RAW 264.7 macrophages Current studies have demonstrated the participation of reactive oxygen species in models of inammation. I. latifolia was investigated as a potential inhibitor of NO production in inammatory reactions. LPS-induced stimulation of RAW 264.7 macrophages led to overproduction of NO, which could be detected and quantied. The results presented in Fig. 5 show that all of the extracts signicantly inhibited NO release and decreased in the order of total avonoids (97%), total saponins (85.25%), total polysaccharides (73.22%) and total alkaloids
Fig. 5 Inhibitory effect on NO production of flavonoids, saponins, polysaccharides and alkaloids extracted from I. latifolia in LPS-induced RAW 264.7 macrophages. Results are mean SD. *P < 0.05, **P < 0.01, statistically significant in comparison with the three others.
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(53.87%) at a concentration of 200 mg mL1. It was noted that total avonoids exhibited the most active anti-inammatory effect among all of the extracts. Even at the lowest concentration (12.5 mg mL1) it also exhibited a better inhibitory effect of above 50%. Lai et al.13 showed that a avonoid compound (formononetin) isolated from the root of Astragalus membranaceus could signicantly inhibit NO production in LPSinduced RAW 264.7 macrophages. In addition, bioactive compounds like saponins have been reported to be benecial in the treatment of chronic inammatory diseases associated with the overproduction of NO. In conclusion, the four main constituents, avonoids, saponins, polysaccharides and alkaloids extracted from I. latifolia exhibited different levels of antioxidant, anticomplement, antitumor and anti-inammatory activities. Total polysaccharides showed an obvious inhibitory effect on the complement system, even stronger than the positive control. The total avonoids displayed signicant DPPH scavenging activity and anti-inammatory effects on LPS-induced RAW 264.7 macrophages. The total saponins showed a better antiproliferative effect against human cervical carcinoma Hela cells, and the total alkaloids exhibited higher reducing power. The results provide useful information on the pharmacological activities of this traditional herbal tea. With the demand for healthy and nutritious plant foods, the nutraceutical market exploring the potential health benets of I. latifolia products is expanding. From a health point of view, kudingcha made from I. latifolia is a useful variety of herbal drinks, due to its various biological activities, suggesting its good potential for the development of dietary supplements. The mechanisms of anticomplement activity of polysaccharides, antioxidation of saponins, and antiproliferation of avonoids are worthy of further research. To the best of our knowledge, this is the rst study to comprehensively evaluate I. latifolia. The results reported here may give researchers insight and guidance in designing future in vivo studies on the health benets associated with the regular intake of I. latifolia.
3. 3.1
Materials and methods Materials
I. latifolia was collected from Wuzhi Mountain, Hainan, China on September 15th, 2012, and was authenticated by Professor Dingyong Wang of the Chemistry department, Guangdong College of Pharmacy, where voucher specimens were kept. Samples were air-dried under shade for one week and ground into a ne powder with a laboratory pulverizer. The dried materials were stored at room temperature in desiccators until use. All other reagents used in the experiments were of analytical grade.
For total avonoids and saponins, the powder (100 g) of I. latifolia was extracted with 80% ethanol for 1 h. The crude extract was evaporated under vacuum to dryness as a dark brown mass. The separation and purication of the two constituents was carried out by using AB-8 macroporous resin eluted with a gradient of ethanol and water (0 : 100, 20 : 80, 50 : 50, 70 : 30, 100 : 0, v/v). The total avonoids fraction (70 : 30 eluates, 6.2 g) and total saponins fraction (50 : 50 eluates, 4.8 g) were obtained according to previous literatures. For the total polysaccharides, the extraction process of polysaccharides (100 g powder) was performed as for the previous method of water extraction and alcohol precipitation.14 The protein was removed from the crude extract and the total polysaccharides sample was obtained (9.6 g). For the total alkaloids, the powder (100 g) of I. latifolia was extracted with an ethanol solution containing a low concentration of hydrochloric acid. The extracts were ltered and concentrated, followed by dissolving in 2% hydrochloric acid, the pH was adjusted to 2–3 and then the insoluble substance was removed. The supernate was extracted with chloroform twice, the chloroform phase was removed, the pH was adjusted to 9–10 using ammonia, and extracted with chloroform until there were no alkaloids in the chloroform phase. The extracts were combined and concentrated to obtain the total alkaloids (2.3 g). The extraction and separation procedure of total avonoids, total saponins, total polysaccharides and total alkaloids from I. latifolia is shown in Fig. 6. 3.3 Determination of the content of the four chemical constituents Modication of the method by Deng et al.15 was conducted to determine the content of the total avonoids at 510 nm. The calibration curve prepared using rutin standard solutions was obtained as Y ¼ 13.113X 0.0177 (R2 ¼ 0.9994), the linear range was 0.01–0.07 mg mL1. The content of the saponins was determined by UV/vis spectrophotometry with ginsenoside Rb1 as the standard examined at 545 nm.15 The regression equation of the standard curve was obtained as Y ¼ 17.925X 0.0498 (R2 ¼ 0.9988), the linear range was 0.05–0.3 mg mL1. The content of the total polysaccharides was conducted using the phenol– sulfuric acid method with D-glucose as the standard at 490 nm,14 which was prepared in sequential concentrations to make a standard curve for the polysaccharide calculation. The regression equation was obtained as Y ¼ 13.821X + 0.0433 (R2 ¼ 0.999), the linear range was 0.01–0.07 mg mL1. Berberine hydrochloride was used to determine the content of the alkaloids as the reference substance that was measured using a colorimetric assay of an acid dye.16 The regression equation of the standard curve was obtained as Y ¼ 7.4484X + 0.0009 (R2 ¼ 0.999), the linear range was 0.04–0.2 mg mL1.
3.2 Extraction of total avonoids, saponins, polysaccharides and alkaloids
3.4
For the purpose of meeting the requirements of the bioactivity assays the four kinds of constituents (avonoids, saponins, polysaccharides and alkaloids) were extracted from I. latifolia in the following ways.
The anticomplement assay through the classical pathway was conducted according to Seo's17 modied method, which was measured by the complement xation test based on complement consumption and the degree of red blood cell lysis by the
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Anticomplement activity
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Fig. 6
The extraction and separation procedure of total flavonoids, total saponins, total polysaccharides and total alkaloids from I. latifolia.
residual complement. The optical density of the supernatant was measured at 405 nm and heparin sodium was used as the positive control. 3.5
Antioxidant assays
3.5.1 Radical scavenging activity of DPPH. The scavenging activity of the DPPH free radical was estimated according to the method of Mu˜ niz-Marquez et al.18 with minor modications. The absorbance of the mixture was measured at 517 nm with a UV-visible spectrophotometer. A decrease of the absorbance indicated an increase of DPPH radical scavenging activity. Analysis of all of the samples was carried out in triplicate with ascorbic acid (Vc) as the positive control. The ability of the samples to scavenge the DPPH free radicals was calculated according to the equation: As Ac DPPH radical scavengingð%Þ ¼ 1 100% Ab Ac where As, Ac and Ab represent the absorbance of the sample, the control and the blank groups, respectively. 3.5.2 Ferric reducing/antioxidant power (FRAP) assay. The ferric reducing/antioxidant power of each sample was measured according to the method of Kapoor et al.19 An increase in antioxidant activity was indicated by increasing absorbance. The absorbance of the mixture solution was measured at 700 nm. Each sample was run in triplicate and averaged. 3.6
Paper
Antitumor activity
The human cervix carcinoma Hela cells were maintained and cultured in tissue culture asks containing DMEM medium
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supplemented with 10% fetal bovine serum, 1% penicillin (100 IU mL1) and streptomycin (100 mg mL1) at 37 C in a humidied incubator with 5% CO2. The effect of each sample on the proliferation of Hela cells was estimated using the MTT assay.20 5-Fluorouracil was used as the positive control. The cell proliferation inhibition rate was calculated using the following formula: Cell proliferation inhibitionð%Þ ¼ 3.7
1
ODsample ODcontrol
100%
Anti-inammatory assays
RAW 264.7 macrophages were cultured in DMEM supplemented with 10% fetal bovine serum, 1% penicillin (100 IU mL1) and streptomycin (100 mg mL1) in a 5% CO2 atmosphere at 37 C. The cells were activated with LPS as in the method described by Hsu et al.21 To assess NO production, the stable conversion product of NO was measured using the Griess reagent and the optical density was determined at 540 nm. The NO inhibition rate was calculated using the following formula: NO inhibitionð%Þ ¼ 3.8
ODLPS ODLPSþsample 100% ODLPS ODblank
Statistical analysis
All of the results are expressed as the means of the three experiments standard deviations (SD). The statistical signicance of the differences between the means was assessed by a one-way analyses of variance (ANOVA). P < 0.05 indicated the presence of a statistically signicant difference and P < 0.01 was considered highly signicant.
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