Anti-Inflammatory Effects of 81 Chinese Herb Extracts and Their Correlation with the Characteristics of Traditional Chinese Medicine

Inducible nitrogen oxide synthase (iNOS) is the primary contributor of the overproduction of nitric oxide and its inhibitors have been actively sought as effective anti-inflammatory agents. In this study, we prepared 70% ethanol extracts from 81 Chinese herbs. These extracts were subsequently evaluated for their effect on nitrogen oxide (NO) production and cell growth in LPS/IFNγ-costimulated and unstimulated murine macrophage RAW264.7 cells by Griess reaction and MTT assay. Extracts of Daphne genkwa Sieb.et Zucc, Caesalpinia sappan L., Iles pubescens Hook.et Arn, Forsythia suspensa (Thunb.) Vahl, Zingiber officinale Rosc, Inula japonica Thunb., and Ligusticum chuanxiong Hort markedly inhibited NO production (inhibition > 90% at 100 μg/mL). Among active extracts (inhibition > 50% at 100 μg/mL), Rubia cordifolia L., Glycyrrhiza glabra L., Iles pubescens Hook.et Arn, Nigella glandulifera Freyn et Sint, Pueraria lobata (Willd.) Ohwi, and Scutellaria barbata D. Don displayed no cytotoxicity to unstimulated RAW246.7 cells while increasing the growth of LPS/IFNγ-costimulated cells. By analyzing the correlation between their activities and their Traditional Chinese Medicine (TCM) characteristics, herbs with pungent flavor displayed potent anti-inflammatory capability. Our study provides a series of potential anti-inflammatory herbs and suggests that herbs with pungent flavor are candidates of effective anti-inflammatory agents.


Introduction
Inflammation is a self-protection mechanism aiming at removing harmful stimuli, including damaged cells, irritants, or pathogens, and beginning the wound repair process. However, inflammation sometimes induces further inflammation, leading to self-perpetuating chronic inflammation that can cause severe cellular injury and tissue damage [1]. Chronic inflammation has been linked to a wide variety of diseases such as atherosclerosis [2], Alzheimer's disease [3], diabetes [4], and carcinogenesis [5][6][7].
Nitric oxide (NO), which is mainly generated by inducible nitric oxide synthase (iNOS) under the inflammatory conditions [8][9][10], plays a key role in each step of the pathological processes during inflammation [11][12][13][14]. Selective inhibitors of iNOS have been shown to be both anti-inflammatory and tissue-protective in various inflammatory animal models [15][16][17] and are thus regarded as promising agents for treating inflammatory diseases. High expression of iNOS can often be detected in human tumors, supporting the notion that chronic inflammation is actively involved in tumor progression [18][19][20][21]. In fact, nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin [22] and tolfenamic acid [23], are currently used for both cancer prevention and treatment [24].
A variety of natural products have been reported to possess anti-inflammatory and anticancer effects in experimental animal models. For example, curcumin has been shown to inhibit cyclooxygenase 2 (COX2) expression and is actually in clinical use as a chemoprevention agent [25][26][27]. Because of the promises in curcumin, extensive efforts have also been exerted to identify compounds capable of targeting inflammatory mediators [28][29][30]. A recent study by Liao et al. investigated the potential association between antioxidation capability and the characteristics of Traditional Chinese Medicine (TCM) in 45 commonly used Chinese 2 Evidence-Based Complementary and Alternative Medicine herbs, in which antioxidation capability of Chinese herbs was found to be correlated with their flavor characteristics [31]. Their findings are very encouraging because it indicates that effective anti-inflammatory agents may potentially be identified from Chinese herbs based on their TCM characteristics.
In our effort to identify effective anti-inflammatory agents, we prepared 70% ethanol extracts from 81 Chinese herbs and subsequently tested their abilities to suppress NO production in murine macrophage RAW264.7 cells costimulated with LPS and IFN . Moreover, we also analyzed the correlation between anti-inflammatory capacity and TCM characteristics among these herbs. We conclude that herbs with pungent flavor are the strongest in their antiinflammatory capability.

Preparation of 70% Ethanol Extracts of Chinese Herbs.
All herbs were obtained from YANG He Tang and Kangqiao Co (Shanghai, China). All 81 herbs chosen for our study have been reported or suggested to have potential antiinflammatory activities by either TCM literatures or current pharmacological reports. Botanical identification of these herbs was performed by Shanghai Institute for Food and Drug Control (SIFDC). To prepare ethanol extracts, 100 g of each dried herbs was sliced and extracted with 1 L of 70% ethanol at 80 ∘ C for three times. Obtained ethanol extracts were evaporated under reduced pressure at temperature 60 ∘ C and stored at −80 ∘ C. Extracts were dissolved with DMSO before use.

Measurement of Nitrite
Production. RAW264.7 cells were plated in 96-well plates (5 × 10 3 cells per well) for overnight and then replenished with FBS-free medium for 10 h followed by adding 100 g/mL herb extracts into each well. Cells were costimulated with 100 ng/mL LPS and 10 U/mL IFN for 24 h, and media were then collected and analyzed for the amount of nitrite, a stable oxidative metabolite and faithful NO indicator, by the Griess reaction as previously described [32]. To do it, 100 L of Griess reagent (0.1% naphthyl-ethylenediamine and 1% sulfanilamide in 5% phosphoric acid) was mixed with 100 L of collected medium in a 96-well plate. Mixture was incubated for 10 min at room temperature and then read at 540 nm. The amount of nitrite was calculated based on a standard curve generated with sodium nitrite. Percent inhibition in NO production was calculated with the formula {[(nitrite with herb extract) − (nitrite without herb extract)]/(nitrite without herb extract)} × 100.

Analysis of Cell
Viability. Cell viability was determined by MTT assay as previously described [33]. Briefly, RAW264.7 cells were incubated with MTT (5 mg/mL in phosphatebuffered saline, pH = 7.4) for 4 h. Formed MTT formazan was solubilized with 50 L of 0.01 M HCl buffer containing 10% SDS and 5% isobutanol. Cell growth was determined by reading plates at 570 nm in a microplate reader. The cell viability of control group is considered as 100%.

Statistical
Analysis. The direction and magnitude of correlation between variables was done using analysis of t-test. values less than 0.05 were considered statistically significant ( * < 0.05).

Effect of Herb Extracts on NO Production and Cell Growth.
With the aid of Griess assay, we analyzed ethanol extracts of 81 herbs for their anti-inflammatory activity. A wide range of inhibition in NO production was observed with these extracts (  (Table 1). Among the extracts that elicited over 50% inhibition in NO production, Rubia cordifolia L., Glycyrrhiza glabra L., Iles pubescens Hook.et Arn, Nigella glandulifera Freyn et Sint, Pueraria lobata (Willd.) Ohwi, and Scutellaria barbata D. Don showed no cytotoxicity to unstimulated RAW264.7 cells while significantly increased the viability of LPS/IFN -stimulated cells (Table 1). However, Daphne genkwa Sieb.et Zucc, which has the strongest inhibitory effect on NO production, was moderately toxic to RAW264.7 cells (Table 1).

Correlation between Anti-Inflammatory Potency and TCM Characteristics of Herbs.
Analyzing the TCM characteristics of 10 herbs that display the strongest inhibitory effect on NO production in LPS/IFN -stimulated RAW264.7 cells, we found that most of them are in the categories of bitter or pungent flavor, warm nature, and lung or liver meridian distributions (Table 2). To correlate the TCM characteristics to anti-inflammatory effect in these herbs, we categorized TCM characteristics of these herbs that were able to abolish 50% of NO production in LPS/IFN -stimulated RAW264.7 cells. Table 3 showed that herbs with greater antiinflammatory effect were distributed in a significantly higher percentage in those characterized as bitter/pungent flavors, warm nature, and liver/lung meridian distributions. These results suggest that anti-inflammatory herbs may possess    common characteristics that are of pungent/bitter flavor, warm nature, and lung/liver meridian.

Correlation between Cell Protective Effect and TCM
Characteristics of Herbs. Chronic inflammation often leads to cell damage and thus agents capable of deterring this process are actively sought. Examining 21 herbs with the TCM characteristic of pungent flavor, we observed that, under the costimulation of LPS and IFN , RAW264.7 cells treated with these herb extracts displayed 90% increase in cell viability (Table 4). Moreover, herbs with pungent flavor also conferred the highest degree of cell protection in LPS/IFNstimulated cells in comparison with herbs with other flavors (Figure 1).

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Discussion
Overproduction of NO due to the elevated iNOS expression has been convincingly linked to the pathogenesis of chronic inflammation and cancer [34]. Hence, agents that can selectively suppress iNOS-generated NO production should be effective to treat chronic inflammation and to prevent cancer. In fact, recent studies demonstrate that selective iNOS inhibitors L-NIL and 1400 W are therapeutically effective as anti-inflammation and anticancer drugs [35,36].
Macrophages play a critical role in regulating inflammation. Macrophages are activated by external stimuli and activated macrophages produce various inflammatory mediators such as NO and reactive oxygen species. Chinese herbs are the rich sources for anti-inflammatory agents and efforts have been made to identify effective components in these herbs [37,38]. Taking  mouse sarcoma S180 in mice [39]. We speculate that anticancer effect of Daphne genkwa Sieb.et Zucc may be functionally associated with its anti-inflammatory capability. In our study, we found that Rubia cordifolia L. and several others decrease LPS/IFN -induced NO production without causing significant cytotoxicity to RAW264.7 cells. These herbs may Evidence-Based Complementary and Alternative Medicine 7 thus be promising candidates as effective drugs to control inflammation and cancer. Although it is currently unclear how these extracts block LPS/IFN-induced NO production in RAW264.7 cells, the finding that Mollugin suppresses the inflammatory response by blocking the Janus kinase-signal transducers and activators of transcription signaling pathway [40] implicates that herbs may target the different steps of the signaling cascade mediating LPS/IFN-induced NO production to exert their anti-inflammatory roles.
Based on the theory of TCM, we classified these 81 herbs according to distinct flavors (pungent, sweet, sour, bitter, astringent, salty, or mild), natures (cold, cool, moderate, warm, or hot), and meridian distributions (liver, kidney, heart, spleen, etc.). Our study showed that the TCM characteristic of flavor correlated very well with the potency to inhibit NO production-pungent flavor is the strongest, bitter is slightly weaker than pungent, sweet flavors is intermediate, and astringent, salty, mild, or sour flavor is weak or not effective. TCM characteristics of nature and meridian distribution are also associated with the potency to inhibit NO production. For instance, higher pe of herbs with the capability to block NO production has the characteristics of warm nature. Characteristics of liver and lung meridians are the major meridian distributions found in herbs whose extracts can block 50% of NO production. Taken together, we reason that TCM characteristics can potentially be very useful to guide the search for effective anti-inflammatory agents in Chinese herbs.
TCM characteristic is a systematic expression of the distinct property elicited by Materia Medica in humans. Theory of flavors in TCM constitutes the core context of Chinese herb usage guidance. In TCM, the characteristic of flavor is the combination of both real taste and curative effect. According to Shen Nong Ben Cao Jing (Shennong's Classic of Material Medica), an important TCM book firstly written on Chinese herbal flavor and property theory, pungent flavor, which is related to lung meridian, can disperse the internal heat with sudorifics which in turn promote the circulation of Qi and blood. Herbs with pungent flavor have actually been used for thousand years in China to invigorate the circulation of blood and break the block of Qi. The fact that inflammation-related diseases are associated with the symptom of Qi and blood blockage may explain the effectiveness of herbs with pungent flavor to suppress inflammation.
Our study was limited to the investigation of 81 herb extracts on their effect on LPS/IFN -induced NO production and cell growth in macrophage RAW264.7 cells. The results generated from this study nevertheless support a close association between modern pharmacology/biomedical science and TCM theory. TCM theory was developed based on thousand years of clinical experience, and the material and pharmacological basis of TCM remains to be explained by the modern biomedical science. We believe that this study has contributed toward this goal.