Effects of processing adjuvants on traditional Chinese herbs.

Processing of Chinese medicines is a pharmaceutical technique that transforms medicinal raw materials into decoction pieces for use in different therapies. Various adjuvants, such as vinegar, wine, honey, and brine, are used in the processing to enhance the efficacy and reduce the toxicity of crude drugs. Proper processing is essential to ensure the quality and safety of traditional Chinese medicines (TCMs). Therefore, sound knowledge of processing principles is crucial to the standardized use of these processing adjuvants and to facilitate the production and clinical use of decoction pieces. Many scientific reports have indicated the synergistic effects of processing mechanisms on the chemistry, pharmacology, and pharmacokinetics of the active ingredients in TCMs. Under certain conditions, adjuvants change the content of active or toxic components in drugs by chemical or physical transformation, increase or decrease drug dissolution, exert their own pharmacological effects, or alter drug pharmacokinetics. This review summarizes various processing methods adopted in the last two decades, and highlights current approaches to identify the effects of processing parameters on TCMs.

1. Introduction

Chinese medicinal processing is a pharmaceutical technique that transforms medicinal raw materials into decoction pieces for use in different therapies based on traditional Chinese medicine (TCM). Processing of crude drugs into decoction pieces is a precious heritage and traditional practice in China, which plays an important role in disease prevention and treatment. The Chinese medicinal materials (CMM) originate from plants, animals, or minerals must undergo appropriate treatments before use as a decoction or other TCM preparations. The Chinese herbal property theory, one of the basic theories in TCM, provides directions for the clinical use of herbs. This theory classifies Chinese herbal properties into four natures, five flavors, ascending or descending, floating or sinking, channel tropism, and toxicity [1]. According to this theory, herbs have special affinities to certain organs and channel systems of the body, and exhibit special effects on diseases of these systems and organs [2]. The potency and toxicity of these herbs may be standardized by processing them according to their characteristics and clinical purpose. Traditional methods, such as stir-frying and steaming, are widely used in herb-processing to prevent exaggerated pharmacological actions, alleviate side effects, modify energy properties (nature, flavor, and channel tropism), mask disagreeable odors, or prolong the shelf-life of crude herbs [3]. Adjuvants are often added to enhance therapeutic effects or minimize drug toxicity, thereby broadening the spectrum of clinical application of the processed drugs. Commonly used adjuvants include vinegar, honey, wine, brine, ginger juice, bran, and rice. Drugs are processed with selected adjuvants based on their specific properties—frying with vinegar adds to the liver-soothing and analgesic effects of drugs, and honey confers Qi-nourishing and lung-moistening effects. Accordingly, the source and quality of adjuvants notably affect the efficacy of processed drugs. The Chinese Pharmacopoeia (2015 edition) lists 117 decoction pieces that are processed with various adjuvants, accounting for 55% of the total number of listed drugs [4]. Compared to simple heat treatment, addition of adjuvants allows tailored enhancement of therapeutic properties of drugs. However, it also complicates the standardization of drug processing methods. Despite being officially described in the Chinese Pharmacopoeia, standards of quality control for processing adjuvants and processed drugs are still lacking. Zhao et al. have discussed the various problems in CMM processing, and emphasized that traditional processing procedures need to be further organized, validated and implemented with scientific understanding to safeguard the quality of decoction pieces [3].

Processing makes TCMs different from other oriental and Western herbal medicines. However, classical TCM theories emphasize on the holistic understanding of diseases and drugs, instead of studying their isolated details. Though classic processing theory and methods have been proven reasonable and reliable in the long-standing clinical practice of TCM, the underlying scientific principles remain largely unknown, affecting the production and use of decoction pieces. Standardization of processing methods, quality control of adjuvants, and related clinical studies were neglected in the past until serious drug misadventures occurred due to improperly processed herbs. Approximately 2396 of 12,354 (19.4%) adverse events, associated with TCM use between 1949 and 2008 in China, are reported to be ascribable to improper processing; besides, over 7000 cases of poisoning due to unprocessed aconitum plants have been reported in the past decade [5,6]. A multi-herb formula is therapeutically more beneficial than a single herb, due to its effects on multiple targets. Synergistic pharmacological effects are often observed with herbal medicines because plant extracts contain compounds that potentiate the action of each other [7]. We speculate that adjuvants similarly act to potentiate the pharmacological effects of drugs. However, identifying their targets at a molecular level is challenging. Fortunately, advanced analytical tools such as MS, NMR, high-throughput screening and omics, offer new avenues to conduct research on TCM at the cellular and molecular level [8]. Significant progress made in this direction in the past two decades necessitates a systematic review to summarize the accumulated knowledge. This review summarizes the commonly used adjuvants and their chemical, pharmacological, and pharmacokinetic mechanisms of synergistic potentiation of drug therapy as well as recent methodological approaches to identify these mechanisms.

2. Mechanisms of interaction between herbs and various processing adjuvants

2.1. Vinegar

Vinegar is consumed as a food condiment worldwide, especially in Chinese cuisines, and also has medicinal uses due to its physiological effects. Different types of vinegars contain organic acids, aldehydes, esters, alcohols, phenols, flavonoids, and ligustrazine [9]. Traditionally, vinegar is widely used in the processing of herbs that soothe the liver, relieve depression, prevent blood stasis, relieve pain, and act as purgatives.

Bupleuri Radix (Chaihu in Chinese), the dried root of Bupleurum falcatum L., is used as a herbal medicine in East Asia to treat influenza, common cold, fever, inflammation, malaria, and menstrual disorders [10]. Vinegar-baked Chaihu has a stronger effect than unprocessed Chaihu on soothing liver and relieving depression. Volatile oils and saikosaponins are the main active ingredients of Chaihu. Baking in vinegar is reported to significantly decrease the content of volatile oils and other antipyretic and anti-inflammatory components, including n-hexanal, n-heptanal, 2-amylfuran, and (E,E)-2,4-sebacic olefin aldehyde [11]. The combined action of heat and acetic acid in vinegar transforms the 13,28-epoxy bridge into a heteroannular diene structure, which accordingly changes saikosaponins a, c, and d to saikosaponins b1 and b2 [12]. Saikosaponins a and d possess notable anti-inflammatory activities, whereas saikosaponin b is a hepatoprotective [13]. In a CCl4-induced liver injury rat model, processed Chaihu exhibited better hepatoprotective effects than raw Chaihu [14]. Additionally, vinegar-baked Chaihu is reported to be a stronger inducer of monoamine neurotransmitters in the depressed mouse brain than crude Chaihu [15]. These findings verify that crude Chaihu may be used for relieving exterior syndromes (cold and fever, white tongue coating, and floating pulse), whereas vinegar-baked Chaihu soothes the liver and relieves depression.

Kansui Radix (Kansui) is the dried root tuber of Euphorbia kansui T. N. Liou ex. T. P. Wang, well-known for treating edema, ascites, and asthma. However, side effects of Kansui, such as inflammation, skin irritation, tumorigenesis, and hepatorenal lesions, limit its clinical use [16]. Ingenol and jatrophane diterpene esters are supposed to be responsible for its toxicity, and these terpenes were found decreased in vinegar-baked Kansui [17]. Transesterification also occurred during baking, leading to the conversion of the toxic 3-acyl ester into the non-toxic 20-acyl ester [18]. Additionally, toxic diterpenes react with organic acids in vinegar to form less water-soluble acylated diterpenes, which diminishes their toxicity [19]. Vinegar-baked Kansui has better effects on relieving ascites, reducing gastrointestinal irritation, and attenuating hepatorenal toxicity than unprocessed [20]. Both Phytolacca Radix (Shanglu), the dried root of Phytolacca acinosa Roxb. or Phytolacca americana L. (Phytolaccaceae), and Genkwa Flos (Yuanhua), the dried flower bud of Daphne genkwa Sieb.et Zucc. (Thymelaeaceae), are diuretics and are processed by vinegar-baking to reduce their toxicities. Vinegar significantly decreases the content of the toxic saponins, EsC and EsB, in Shanglu and increases the content of the therapeutically active saponin, EsA [21-24]. Furthermore, vinegar-baking decreases the levels of the toxic benzoyl-diterpenes, yuanhuacin and genkwadaphnin, practically eliminating the toxicity of Yuanhua [25,26].

In addition to its detoxifying effects, vinegar increases the water solubility of active substances in herbs, thereby increasing their pharmacological activities. Corydalis Rhizoma (Yanhusuo), the dried tuber of Corydalis yanhusuo (Papaveraceae), promotes blood circulation and relieves pain. Vinegar-baking potentiates these effects as the acetic acid in vinegar solubilizes the free alkaloids of Yanhusuo [27,28].

In general, acetic acid in vinegar along with the high temperature of baking promotes complex chemical reactions, such as pyrolysis, hydrolysis, esterification, and salification. Moreover, various pharmacological effects of vinegar per se have been explored, and the improved therapeutic effect of vinegar-processed drugs could be due to several mechanisms of actions [29]. Further research works are needed to support the reports of the benefits of vinegar on drug processing.

2.2. Honey

Herbs are usually fried with honey to improve their Qi-nourishing and lung-moistening effects. Ephedrae Herba (Mahuang), the dried stem of Ephedra sinica Stapf (Ephedraceae), is notably used for diaphoresis and relieving exterior syndromes in Chinese medicine. Frying in honey reduces the volatile oil content, responsible for diaphoresis, whereas the contents of ephedrine and pseudoephedrine only decrease slightly [30,31]. Thus, the cough-relieving and anti-asthmatic effects become relatively prominent when the effect of diaphoresis is weakened [32]. Consequently, Mahuang is traditionally processed by frying in honey for its effects of relieving cough and asthma. Nevertheless, honey may also possess its own antitussive effects, which might be majorly responsible for synergistically increasing the cough-relieving properties of Mahuang, rather than its effects on volatile oil composition. This is evident in many cultures, where honey is used as an alternative remedy to treat the symptoms of upper respiratory tract infections (URIs), including cough [33]. Honey-frying is also used to process other lung-moistening and antitussive herbs, such as Peucedani Radix (Qianhu), Farfarae Flos (Kuandonghua), Eriobotryae Folium (Pipaye), and Stemoae Radix (Baibu) [34-40].

Due to its high glucose and fructose contents, honey boosts nonspecific immunity and macrophage phagocytosis in vivo [41]. TCM theory proposes that Qi-tonifying effects on the body are associated with improved immune function, including the activation of T and B lymphocytes and regulation of innate immunity [42]. Therefore, licorice [the root of Glycyrrhiza glabra L. (Fabaceae)] and other tonic herbs are fried with honey to increase their spleen-stimulating and Qi-enhancing effects. A study showed that, compared with simply fried product, honey-frying does not alter the composition of licorice; however, honey-fried licorice aids weight gain, prevents fatigue, and improves the spleen and thymus indices in mice [43].

Several foods and drugs either induce or inhibit CYP activity to alter drug pharmacokinetics and pharmacodynamics [44]. Animal studies have shown that multiple doses of honey induce CYP3A4 but inhibit CYP2C9 activity, whereas a clinical trial revealed that honey from south India only induces CYP3A4 activity in healthy volunteers [45]. Flavones and polyphenols in honey were shown to be responsible for the CYP3A4 induction; however, further studies on the therapeutic effects of honey-processed herbs are required [46]. It is worth noting that herbs are usually processed with refined honey, which differs from raw honey in its chemical and pharmacological characteristics. Because the source of honey has a great influence on its composition and herb-processing effects, mentioning the source is crucial.

2.3. Wine

Wine is popularly used in herb processing. Ancient literature reports that wine changes herbal nature by promoting the upward direction and cleaning the upper-energizer heat, thereby enhancing the efficacy of herbs for invigorating the blood [47]. Alcohol is a good organic solvent that dissolves most water-soluble or insoluble substances in herbs. Due to its good permeability, it enters plant tissues to promote displacement, diffusion, and dissolution of the phytoconstituents. Rhubarb, the root of Rheum palmatum L., is a potent purgative, and only a small dose for a short treatment period is recommended [48].

To moderate its potency and toxicity, rhubarb is fried (Shudahuang) or steamed (Jiudahuang) with yellow wine to prepare wine-processed rhubarb. Rhubarb, processed in these ways, exhibits lower purgative and higher anti-blood stasis effects than raw rhubarb [49-51]. Wine decreases conjugated anthraquinone content, and dramatically increases free anthraquinone content, causing mild diarrhea and toxicity [52,53]. It may be hypothesized that heat treatment decomposes the conjugated anthraquinones, and wine promotes the dissolution of active ingredients, thereby reducing the toxicity and enhancing the efficacy of rhubarb [54]. Radix Scutellariae (Huangqin), the dried root of Scutellariae baicalensis Georgi, is a well-known TCM used for the treatment of inflammation, ulcers, and hepatitis. Flavonoids, such as baicalin, are responsible for the pharmacological effects of this herb [55]. Pharmacokinetic parameters, such as Cmax and AUC0–t, of some flavonoids remarkably increased in the upper-energizer tissues (lung and heart) but decreased in the middle-and lower-energizer tissues (spleen, liver, and kidney) of rats administered wine-processed Huangqin compared with those in rats administered unprocessed Huangqin [56]. Tissue distribution of flavonoids agrees with the ascending and descending theory, indicating that wine has the ability to “induce medicine upward” and concentrate drug components on upper-energizer tissues [47]. Wine processing along with heat treatment increases the dissolution of flavonoids in Huangqin, by increasing the total surface area, fractal dimension, and mesopores [57]. Baicalin and wogonoside are the main active flavonoid glycosides of Huangqin that tend to be hydrolyzed by some enzymes in crude herbs. Wine-processing could deactivate the enzymes to reduce their loss, thereby improve the antibacterial and anti-inflammatory effects [58].

Moreover, alcohol, being a natural preservative, allows the storage of medicinal liquor for months or years without deterioration. Wine also masks unpleasant odors and increases palatability. Drugs of animal origin, such as those obtained from the Zaocys (Wushaoshe) or Agkistrodon (Qishe) genera of snakes, Pheretima (Dilong) and geckos (Gejie), have a foul reptilian odor due to trimethylamine. This gut microbial metabolite of choline evaporates with ethanol when such drugs are fried with wine [59].

2.4. Brine

Brine is a highly-concentrated solution of salt, especially sodium chloride. According to TCM theory, herbs are processed by frying or steaming after moistening with brine to conduct the drug to the kidney meridian and improve the curative effect on lower-energizer syndrome. Frying the bark of Eucommia ulmoides (Duzhong) with brine improves its kidney-tonifying function and alleviates osteoporosis [60]. Salt-frying promotes the absorption and bioavailability of geniposide in Duzhong, even its absolute content decreased sharply after processing [61]. Presence of sodium and chloride ions plausibly improves its intestinal absorption [62]. Psoraleae Fructus (Buguzhi) is the ripe fruit of Psoralea corylifolia L. (Fulse). Salt-frying increases the contents of psoralen and isopsoralen coumarins in Buguzhi, increasing their intestinal absorption in rats [63,64]. Salt-processing of Buguzhi is also reported to increase the distribution of psoralen and isopsoralen to generative organs, the heart, and the spleen. Moreover, their distribution to generative organs (lower-energizer) is significantly higher than that to the heart and spleen, thereby directing drugs to the kidney meridian [65]. Although salt-processing alters the pharmacokinetics of both these coumarins, the change in chemical composition is believed to occur by heating. Whether salt participates directly in chemical reactions is yet to be proven. Recently, salt was reported to affect the cell wall permeability of plant tissues, which makes them more susceptible to rupture during processing and help dissolve the phytoconstituents in the decoction. Moreover, by changing the ionic composition of the reaction system, brine processing influences the chemical reactions in herbs [66]. Dietary salt has been proved to affect drug disposition by modulating sympathetic activity and the intestinal expression of CYP3A4 or P-glycoprotein, resulting in a local alteration of drug-metabolizing activity and drug transport in intestine [67,68].

2.5. Herbal juice

The processing of crude drugs with juices of herbs is an important part of CMM processing. Ginger and licorice, two herbs frequently appear in TCM formula, are commonly served as adjuvants in drug processing. In addition to modifying the properties of other drugs by chemical or physical interactions, these adjuvants exert their own pharmacological activities via synergistic action on multiple pathways.

Rhizoma Coptis (Huanglian), the dried rhizome of Coptis chinensis Franch, is a classic heat-clearing and detoxifying herb that is bitter-cold in nature. After processing with ginger juice, its effect on heat-clearing and the stomach meridian is strengthened. Huanglian has a good anti-bacterial property and serves as a remedy for intestinal infections. An in vitro study confirmed that its antibacterial effect was enhanced in combination with ginger juice, which also exerts some antibacterial effect [69]. This combination inhibits ethanol-induced damage to the gastric mucosa in rats, owing to the synergistic inhibition of pro-inflammatory cytokine release [70]. Danfupian is obtained by processing desalted aconite [the lateral root of Aconitum carmichaeli Debx. (Ranunculaceae)] with juices of licorice and black bean (semen of Glycine max). This processing transforms toxic diester alkaloids in aconite into less toxic monoester alkaloids through transesterification with fatty acids of licorice. Moreover, these diester alkaloids form insoluble precipitates with glycyrrhizic acid, thereby reducing aconite toxicity [71].

2.6. Oil

Epimedii Folium (Yinyanghuo), the dried leaf of Epimediium brevicornu Maxim (Berberidaceae), is used to treat erectile dysfunction in East Asia. However, poor solubility of the active flavonoid components results in poor bioavailability and limited clinical efficacy of Yinyanghuo [72]. According to traditional processing methods, frying with suet oil strengthens its effect of warming kidney and enhancing yang, which has been proved in hydrocortisone-induced kidney-yang deficiency rat models [73]. The synergy between heating and suet oil in the processing of Yinyanghuo perfectly solves the problem of absorption. Heating initiates the deglycation of flavonoid glycosides in Yinyanghuo to produce easily absorbable bioactive flavonoids, such as icariin and baohuoside. Moreover, suet oil along with sodium deoxycholate, an endogenous bile salt, forms self-assembled nanomicelles to promote carrier-mediated absorption [74,75].

2.7. Alum/lime

Pinellia Rhizoma (Banxia), the dried tuber of Pinellia ternata (Thunb.) Breit. (Araceae), improves Qi, alleviates external pain and swelling, acts as an anti-emetic, and relieves stuffiness. Sharp raphides of calcium oxalate and an agglutinin present in crude Banxia cause toxicity that manifests as mucosal irritation, leading to tingling sensations on the tongue, tongue swelling, aphonia, vomiting, and diarrhea [76]. Raphides directly pierce the mucous membranes and lead to cell damage. The pro-inflammatory agglutinin stimulates the release of inflammatory mediators and causes pain. Calcium oxalate is water-insoluble at neutral pH; however, soaking in alum/lime water, solubilizes these raphides. It also degrades the toxic agglutinin and diminishes the irritation caused by crude Banxia [77]. To enhance its therapeutic effects, ginger or licorice is used as a secondary adjuvant. Although ginger does not neutralize Banxia toxicity, it acts as an immunosuppressant to reduce inflammatory response [78]. Multi-adjuvant processing also reduces the toxicity of Arisaematis Rhizoma (Tiannanxing) and Typhonii Rhizoma (Baifuzi) [79]. Use of multiple adjuvants in combination synergistically enhances the chemical, physical, and pharmacological aspects of herbs, which is a subtle application of compatibility theory of TCM in herbal processing.

2.8. Solid adjuvants

Solid materials, such as sand, bran, rice, and gecko shell powder, are commonly used to assist stir-frying. Although not chemically involved, these solid adjuvants physically interact with treatment agents. Pangolin scales (Manis Squama) become crisp and easy to decoct after scalding by hot sand [80]. Upon frying, wheat bran absorbs the volatile oils of Rhizoma Atractylodis Macrocephalae (Baizhu) and reduces its dryness [81]. Rice is added while stir-frying the Chinese blister beetle, Mylabris phalerata (Meloidae), to keep it at a moderate heat and prevent excessive charring. Furthermore, the added rice absorbs cantharidin, a toxic sesquiterpene secreted by the beetle [82].

2.9. Overview of CMMs processing research

Multifarious materials appeared and phased out over the long course of development of Chinese medicines. At present, over 14 types of adjuvants are recorded in the Chinese pharmacopoeia and applied to more than 100 kinds of drugs. Various commonly used processing adjuvants along with their chemical, pharmacological, toxicological and pharmacokinetic modifications of the processed drugs are listed in Table 1. A diagram of general effects of adjuvants in CMM processing is shown in Fig. 1.

Table 1

Interaction between herbs and processing adjuvants as reported in the literatures.

Herb processed	Adjuvant	Type of interaction		Clinical outcomes	Ref.
		Chemistry	Pharmacology/Toxicology/Pharmacokinetics
Bupleuri Radix	vinegar	(↑) saikosaponins b1 and b2;(↓) n-hexanal, n-heptanal, 2-pentylfuran, (E,E)-2,4-decadienal, and saikosaponins a, c, d	(↑) estrogen regulation, hepatoprotection, choleretic effect, and anti-depressant and analgesic effects1;(↓) anti-inflammatory effects1;(−) induction of CYP2C9 and CYP2C191;(↑) Cmax and AUC0–t of saikosaponins b1 and b21;(↓) Cmax and AUC0–t of saikosaponins a, b3, and d1	Improves liver soothing and choleretic effects, weakens exterior syndrome relieving and antipyretic effects	[10–15,83–88]
Kansui Radix	vinegar	(↓) toxic diterpenoids (kansuinine A, B, D, kansuiphorin C, euphol, etc.) and triterpenoids (euphol, kansenone, epi-kansenone, 11-oxo-kansenonol)	(↑) diuretic effect1;(↓) hepatotoxicity, gastrointestinal toxicity, carcinogenesis, purgation1; inflammation, skin irritation2	Reduces toxicity, improves diuretic action	[16–20,89–93]
Schisandrae Chinensis Fructus	vinegar	(↑) lignans (schisandrin, gomisin D, schisantherin A), and protocatechuic acid;(↓) lignans (schisantherins B, C, D, 6-O-benzoylgomisin O), neokadsuranic acid, and volatile oil	(↑) antidiarrheal, sedative, hypnotic, anti-lipid peroxidation, and immunity enhancement effects1;(↑) CYP3A4 induction and inhibit CYP1A2 activity1;(↑) Tmax, MRT0–t, AUC0–t, Re, and Ce of schisantherin and deoxyschisandrin in liver, and causes biliary excretion of metabolites with acute liver injury1	Leads drug to the liver meridian, improves astringent action	[94–100]
	wine	(↑) lignans (Gomisin D, T, schisandrins A, B, and C);(↓) lignans (schisantherins B, C, and D), and neokadsuranic acid	(↑) increases murine splenic lymphocyte proliferation2; renoprotection1;	Majors in warming kidney and strengthening yang
Schisandrae Sphenantherae Fructus	vinegar	(↓) volatile oil	(↑) hepatoprotection1;(↑)Tmax, MRT0–t, AUC0–t, Re, and Ce of schisantherin and deoxyschisandrin in liver1	Leads drug to the liver meridian	[101,102]
Olibanum	vinegar	(↑) α-boswellic acid, 11-keto-β-boswellic acid, and 11-keto-β-acetyl- boswellic acid;(↓) β-boswellic acid and 3-acetyl-β-boswellic acid	(↑) anti-platelet adherence, anti-inflammation and anticoagulation1(↑) Cmax, AUC, T1/2, and MRT1	Promotes blood circulation to treat blood stasis, reduces digestive tract irritation, eases pulverization	[103–106]
Cyperi Rhizoma	vinegar	(↑) cyperotundone and luteolin;(↓) nootkatone and α-cyperone	(↑) analgesic and anti-inflammatory effects, intestinal propulsion rate1	Soothes the liver to relieve pain, and relieves dyspepsia	[107–109]
Corydalis Rhizoma	vinegar	(↑) tetrahydropalmatine (THP), protopine, palmatine(↓) α-allocryptopine, coptisine, palmatine, and dehydrocorydaline (DHC)	(↑) analgesic and spasmolytic effects1;(↑) THP level in the rat plasma and liver1; Tmax of DHC in the heart, kidney, cerebrum, cerebellum, brain stem, and striatum1; and Tmax of protopine in brain1;(↓) MRT of DHC in the spleen, lung, cerebrum, and diencephalon; MRT of protopine in the heart, spleen, and kidney1	Promotes Qi circulation to relieve pain	[27,28,110,111]
	wine	(↑) tetrahydrocolumbamine, THP, corydaline, tetrahydroberberine, and tetrahydrocoptisine;(↓) protopine, α-allocryptopine, coptisine, palmitine, berberine, and DHC	(↑) Tmax of tetrahydroberberine in all the tissues1;(↓) Tmax of protopine and DHC in the liver and spleen1; and Tmax of protopine in the lungs1	Promotes blood circulation and treats blood stasis
Curcumae Rhizoma	vinegar	(↓) curdione, germacrone, bisdemethoxycurcumin, demethoxycurcumin, and curcumin	(↑) anti-platelet aggregation, anticoagulation, hepatoprotective, anti-inflammatory, analgesic1; and anti-tumor effects2;(+) inhibition on CYP1A2 and CYP2E1, and induction of CYP3A41	Leads the drug to the liver meridian, treats blood stasis, and relieves pain	[112–116]
Phytolaccae Radix	vinegar	(↑) esculentoside A;(↓) esculentosides B and C	(↑) diuretic effect1;(↓) conjunctival irritation, gastric mucosal irritation, intestinal edema, diarrhea, and purgation1	Reduces toxicity, moderates potent diuretic action, and majors in relieving edema	[21–24]
Strychni Semen	vinegar	(↓) strychnine and brucine	(↑) anti-inflammatory and analgesic effects1;(↓) LD501	Reduces toxicity	[117–119]
Genkwa Flos	vinegar	(↑) kaempherol, apigenin, 3′-hydroxygenkwanin, genkwanin, genkwanine N, and genkwadaphnin(↓) luteolin, isodaphnoretin, yuanhuacine, genkwadaphnin, and genkwanin-5-O-β-D-primeveroside	(↑) diuretic effect1;(↓) toxicity1	Reduces toxicity and improves diuretic effect	[25,26]
Radix Paeoniae Alba	vinegar	(↑) albiflorin(↓) paeoniflorin	(↑) analgesic and sedative effects1	Leads the drug to the liver meridian, nourishes the blood, soothes the liver, and relieves depression	[120–122]
Ephedrae Herba	honey	(↓) ephedrine, pseudo-ephedrine, and volatile oil	(↑) anti-asthmatic effect1;(↓) diaphoresis1	Moderates diaphoresis, major in freeing lung and relieves asthma	[30–32,123]
Peucedani Radix	honey	(↑) praeruptorins A, B, and E	(↑) antitussive, expectorant, and anti-asthmatic effects1	Moistens the lung to stop cough	[34,35]
Farfarae Flos	honey	(↑) rutin, isoferulic acid, and tussilagone;(↓) chlorogenic acid, apigenin, and senkirkin	(↑) antitussive and expectorant effects1;(↓) toxicity2;	Moistens the lung to stop cough	[36,124]
Stemonae Radix	honey	(↓) stenine, oxystemoninine, stemonine, N-oxytuberostemonine, and tuberostemonine H	(↑) antitussive and anti-asthmatic actions1;(↓) acute toxicity1	Moistens the lung to stop cough, moderates gastric irritation	[38,40]
Glycyrrhizae Radix et Rhizoma	honey	(↑) 5-HMF;(↓) glycyrrhizin, liquiritin, liquiritin apioside, licuraside, and isoliquiritin	(↑) immunity1;(↓) antitussive and expectorant effects, detoxication, CYP3A4 induction1	Tonifies the spleen and stomach	[43,125–128]
Astragali Radix	honey	(↑) astragalosides I, III, and IV, calycosin-7-O-β-d-glucoside, and formononetin-7-O-β-D-glucoside;(↓) calycosin, formononetin, and astragaloside IV	(↑) anti-fatigue effect and anoxia endurance1	Exerts center-supplementing and Qi-boosting effects	[129–131,206]
Cimicifugae Rhizoma	honey	(↑) caffeic acid, ferulic acid and isoferulic acid	(↑) analgesic and sedative effects1	Moderates diaphoresis, majors in elevating spleenyang	[132,133]
Aristolochiae Fructus	honey	(↓) aristolochic acids I, II, C, and D	(↓) nephrotoxicity1	Moderates the bitter-cold nature, moistens the lung to stop cough, modifies the taste, and reduces vomiting	[134,135]
Polygalae Radix	honey	(↑) sibiricose A6, glomeratose A,(↓) polygalacic acid, senegenin, onjisaponin B, tenuifoliside B sibiricose A5, and 3, 6′-disinapoyl sucrose	(↑) LD50, antitussive and expectorant effects1;(↓) inhibition on gastrointestinal motility and digestive function1	Improves cough relieving effect and dissipates phlegm	[136–143]
	licorice	(↑) Tenuifolin, polygalacic acid, glomeratose A, senegenin, organic acids (sinapic acid, pcoumaric acid, ferulic acid, benzoic acid, cinnamic acid);(↓) sibiricose A5 and A6; tenuifoliside B, and 3, 6′-disinapoyl sucrose	(↑) anti-alcoholism effect1;(↓) inhibition on gastrointestinal motility and digestive function1	Moderates dryness, eliminates tongue numbing and throat irritation; tranquilizes the mind and promotes intelligence
Rhei Radix et Rhizoma	wine	(↑) emodin, rhein, aloe-emodin, and gallic acid(↓) physcion, chrysophanol, and sennosides A and B	(↑) antipyretic and anticoagulant effects, permeability of blood–brain barrier, ulcer healing, and embryotoxicity1;(↓) purgative effect, and hepatorenal toxicity1;(↓) anthraquinone metabolites1	Moderates bitter-cold nature, majors in clearing virulent pyropathogen of upper energizer	[48–54,144–146]
Radix Scutellariae	wine	(↑) baicalin, oroxylin A-7-O-glucuronide, and wogonoside	(↑) antibacterial2; antiviral, analgesic, and antiinflammatory effects1;(↓) antioxidant effect2;(↑) Cmax and AUC(0–t) of major flavonoids in the lungs1;(↓) Cmax and AUC(0–t) of major flavonoids in the kidneys1	Leads the drug upwards, clears the lung heat and damp heat of limbs	[55–58]
Salvia Miltiorrhiza Radix et Rhizoma	wine	(↑) dihydrotanshinone I and tanshinone I;(↓) tanshinone IIB, cryptotanshinone, salvianolic acid B, neotanshinone B, tanshinone IIA, miltirone, and protocatechuic aldehyde	(↑)anticoagulation activity1; α-glucosidase inhibition, antimicrobial, and antioxidant activity2	Moderates cold nature, activates blood circulation to treat blood stasis, regulates menstruation, and relieves pain	[50,147–149]
Achyranthis Bidentatae Radix	wine	(↑) benzyl glucoside, polypodine B, βecdysterone, and ginsenoside Ro;(↓) zingibroside R1, bidentatoside I, and chikusetsusaponin IV	(↑) analgesic and anti-inflammatory effects, immunity, and hemorheology1;(↓) EBV-EA activation2;	Tonifies the liver and kidney, strengthens the bones and muscles, treats blood stasis, and relieves pain	[150–153]
	salt	(↑) benzyl glucoside, polypodine B, βecdysterone, achyranthesterone A, βecdysterone, and inokosterone;(↓) zingibroside R1, ginsenoside Ro, bidentatoside I, and chikusetsusaponin IV	(↑) EBV-EA activation1;(↓) LD501	Leads the drug to the liver meridian, tonifies the liver and kidney, strengthens the physique, promotes diuresis, and relieves stranguria
Corni Fructus	wine	(↑) gallic acid, sweroside, cornin, 5- hydroxymethylfufural, 7α-O-ethylmorroniside, and 7β-O–ethylmorroniside;(↓) cornuside, morroniside, and loganin	(↑) antioxidant activity2; immunity enhancement, and protection against acute liver injury1;(↓) α-glucosidase inhibition activity2; hypoglycemic activity1;(↑) T1/2, AUC0–t, and Cmax of morroniside and loganin1	Nourishes yin and tonifies the kidney	[154–157]
Polygonati Rhizoma	wine	(+) DDMP and 5-HMF;(↑) low molecular weight saccharides;(↓) diosgenin	(↑) antioxidant activity2; immunity enhancement1	Reduces irritation, nourishes yin, and tonifies the kidney	[158–161]
Coptidis Rhizoma	wine	(↑) berberubine, noroxyhydrastinine, and worenine;(↓) magnoflorine, jatrorrhizine, columbamine, epiberberine, coptisine, plamatine, and berberine	(↑) anti-bacterial and improvement in insulin resistance effects2; hypoglycemic activity, and sedative–hypnotic activity1;(↓) antioxidant activity2;(↑) Cmax of coptisine and 8-oxocoptisine, AUC0–t of coptisine, palmatine, and 8-oxocoptisine1	Improves drug ascending, moderates cold nature, and majors in clear heat of up-energizer	[69,70,162–166]
	ginger	(↓) berberine, plamatine, epiberberine and coptisine	(↑) Na/K-ATPase activity, and gastric mucosal protection1; antibacterial effect2	Moderates bitter-cold nature and arrests vomiting
	Euodiae Fructus	(↓) berberine, epiberberine and coptisine	(↑) anti-bacteria2; anti-diabetes, anti-gastric ulcer1	Moderates bitter-cold nature, and majors in clearing stagnated heat in the liver and stomach
Eucommiae Cortex	salt	(↑) coniferylaldehyde, pinoresinol, epipinoresinol, medioresinol, and medioresinol;(↓) genipin, geniposide, geniposidic acid, caffeic acid, chlorogenic acid, quercetin, and pinoresinol diglucoside	(↑) prevents osteoporosis1;(↑) Cmax and AUC of geniposidic acid1	Leads the drug to the kidney meridian, and tonifies the liver and kidney	[60–62,66]
Psoraleae Fructus	salt	(↑) psoralen, isopsoralen, bavachin, corylin, isobavachalcone, and bavachalcone;(↓) bavachromanol, bakuchiol, and bavachinin A	(↑) anti-diarrheal1; antioxidant, anti-osteoporosis, α-glucosidase inhibitory activities2;(↓) toxicity2;(↑) Ka of psoralen and isopsoralen2	Increases drug disposition into kidney, promotes warming kidney, and activates yang	[63–65,167–172]
Anemarrhenae Rhizoma	salt	(↑) timosaponin BIII;(↓) timosaponins I, E1, and BII	(↑) α-glucosidase inhibition, hypoglycemic effect2; anti-hyperthyroidism, and laxation1;(↑) Cmax, AUC, and MRT of neomangiferin1	Leads the drug to the kidney meridian, nourishes yin to reduce pathogenic fire	[173–178]
Morindae Officinalis Radix	salt	(↑) monotropein	(↑) anti-inflammatory and anti-hypoxic effects; renoprotection, and improves thyroid dysfunction1;(↑) distribution of monotropein in the kidney, liver, and spleen1	Reinforces the kidney-yang	[179–182]
	licorice	(↑) monotropein	(↑) distribution of monotropein in the spleen1;(↓) distribution of monotropein in the kidney and liver1	Majors in tonifying the kidney-yang
Phellodendri Chinensis Cortex	salt	(↑) berberubine;(↓) limonin, obacunone, berberin, plamatine, and jateorizine	(↑) anti-hyperthyroidism1;(↓) antioxidant effect2; weight loss, and gastrointestinal dysfunction1;(↑) CYP3A4 induction1;(−) CYP1A2 inhibition1	Leads the drug to the kidney meridian; moderates bitterness and dryness; nourishes yin; and purges fire	[183–187]
	wine	(↓) berberin, plamatine, jateorizine, limonin, and obacunone	(↑) anti-oxidation2; bacteriostatic1;(↓) anti-hyperthyroidism and gastrointestinal dysfunction1;(+) CYP2C9 induction1;(↑) CYP3A4 induction1;(↓) CYP1A2 inhibition1	Weakens the bitter-cold nature, leads the drug upward, and majors in clearing heat in upenergizer
Alismatis Rhizoma	salt	(↑) alisol A, B and alisol A 24-acetate;(↓) alisol B 23-acetate	(↑) diuretic, anti-inflammatory, immunomodulation1	Nourishes yin and promotes diuresis	[188–190]
Magnoliae Officinalis cortex	ginger	(↑) magnolol and honokiol	(↑) analgesic and anti-inflammatory effects, bacteriostasis and gastrointestinal motility1;(↓) irritation1	Eliminates throat irritation and promotes stomach harmonization	[191–193]
Polygoni Multiflori Radix	black bean	(↑) emodin, chrysophanol, and physcion;(↓) stilbene glycoside, catechin, 2,3,4′,5- tetrahydroxystilbene 2-O-β-D-glucoside, and emodin-8-O-β-D-glucoside	(↑) improve hematopoietic and hemorheological function1;(↓) hepatotoxicity, laxation effects1; antioxidant2	Improves tonifying kidney, essence, and blood; blackening hair, strengthens physique, and lowers toxicity	[194–197]
Aconiti Lateralis Radix Praeparata	salt, licorice, and black bean	(↓) diester alkaloids	(↑) analgesic and anti-inflammatory effects1;(↓) acute toxicity1	Reduces toxicity	[71,198]
Epimedii Folium	Suet oil	(↑) icariin and baohuoside	(↑) anti-prostatic hyperplastic and HPAT functions1;(↑) Papp, Tmax, Cmax, and AUC of icariside I1	Improves warming kidney and strengthening yang	[199–201]
Pinelliae Rhizoma	alum	(↓) calcium oxalate raphides, total protein, guanosine and uridine	(↑) anti-inflammatory and antitussive effects1;(↓) pro-inflammatory effect1,2	Majors in removing dampness to reduce phlegm	[76–78,202–205]
	alum/ginger		(↑) antitussive, expectorant, analgesic, antiemetic and anti-inflammatory effects1;(↓) emesis, embryo toxicity1 and pro-inflammatory effect1,2	Improves downbear counterflow and prevents vomiting, majors in warming middle, and dissipating phlegm
	licorice/ lime		(↑) antitussive and sedative effects1;(↓) pro-inflammatory effect2	Dispels cold phlegm; harmonizes the liver and spleen
Typhonii Rhizoma	alum/ginger	(↑) 5-HMF and bis (5-formylfurfuryl) ether;(↓) calcium oxalate raphides	(↑) sedative, anticonvulsant, analgesic, and antiinflammatory effects1;(↓) irritation and toxicity1	Reduces toxicity, eliminates mucosal irritation, and dispels wind-phlegm	[205–209]
Atractylodis Macrocephalae Rhizoma	bran	(↑) atractylenolides I, II, and III;(↓) atractylon	(↓) peristalsis of the small intestine1	Moderates dryness, invigorates the spleen, and harmonizes the stomach	[81,210]
Mylabris	rice	(↓) cantharidin	(↑) anti-tumor activity1;(↓) LD50, hepatic, renal, and gastrointestinal toxicity1	Reduces toxicity	[82]

Abbreviations (↑) increases; (↓) decreases; (+) new appeared; (−) disappeared; 1 in vivo; 2 in vitro.

Fig. 1

General effects of adjuvants in CMM processing.

3. Analytical tools for evaluating the effects of processing on herbs

3.1. Chemical characterization by chromatographic or spectroscopic methods

Herbal medicines rely on multiple components to exert pharmacological effects. Accordingly, the isolation and identification of individual components in most herbs remain a great challenge due to their structural diversity and complexity. Novel chromatographic and high-resolution tandem mass spectrometric methods facilitate the structural characterization of complex compounds in TCM with improved accuracy and sensitivity [211]. Astragali Radix (Huangqi), the dried root of Astragalus propinquus (Fabaceae), is widely used as a tonic in TCM. It is processed with honey to yield a product with reduced side effects and improved efficacy in tonifying Qi. To identify the pharmacological benefits of processing with honey, 35 compounds in crude and honey-processed Huangqi were detected and identified in a 23-min run using UPLC/ESI-Q-TOF-MS. Quantitative analysis revealed that honey processing reduces isoflavonoid contents and increases saponin contents. Since Astragalus saponins are known to regulate the immune functions of macrophages, an increase in their contents translates into enhanced immunity [212]. Being a quick, label-free, and nondestructive analytical technique, infrared spectroscopy allows the in situ monitoring of the changes in herb composition during processing. Thermal processing of Gardeniae Fructus (Zhizi), the ripe fruit of Gardenia jasminnoides Ellis (Rubiaceae), decreases its organic acid content, as studied by thermogravimetry-infrared spectroscopy. This was found to reduce its toxic effects on the intestines and stomach. Stir-baking to yellow (125–145 °C) remarkably reduces organic acid content, whereas most iridoids, such as geniposide, remain unaffected by baking at this temperature. Consequently, the pathogenic heat clearing activity of this fruit is retained. However, stir-baking to scorched (165–190 °C) destroys most iridoids so that tannins in the fruit exert hemostatic effects [213]. Other spectroscopic approaches, such as NMR, XRD, and Raman spectroscopy, in combination with appropriate chemometric methods, also aid in interpreting the mechanistic alteration of pharmacological effects by adjuvants and the dynamic monitoring of TCM processing [57,214,215].

3.2. Dose-effect correlation analysis

Because processing alters the chemical composition and efficacy of drugs, it is necessary to evaluate the quality of processed products by specific components in light of their pharmacological effects. Chemometric methods, such as correlation analysis and principal component analysis (PCA), facilitate the evaluation of the chemical profile in relation to the pharmacological and toxicological profile of TCMs [216]. Chronic treatment with rhubarb is reported to cause hepatorenal lesions, whereas wine processing enhances its anti-blood stasis effects and reduces its toxicity [8]. A previous bioactivity assay reported that wine-treated rhubarb has a higher potency than crude and charred rhubarb. Furthermore, UPLC fingerprint analysis identified that rhein-8-O-β-D-glucoside, emodin-8-O-β-D-glucoside, and rhein strongly inhibit platelet aggregation [217]. Based on canonical correlation analysis (CCA) to investigate the correlation between toxicity and chemical composition of processed rhubarb, it was reported that the reduced toxicity in processed rhubarb was due to the decrease in free and conjugated anthraquinone (aloe-emodin and physcion) and tannin concentrations [218]. Similarly, UPLC fingerprinting, microcalorimetry, and CCA were combined to explore the different effects of aconite and its processed products on energy metabolism. The results revealed that mesaconitine, benzoylaconitine, and benzoyl-hypacoitine affect mitochondrial energy metabolism in rats [219].

3.3. Metabolomics

Metabolomics involves the global profiling of endogenous metabolites and dynamic responses to both endogenous and exogenous factors to identify complex interactions among biological systems, drugs, and diseases [220]. Complex chemical compositions of TCMs demand a metabolomic approach to identify their diverse metabolic pathways in relation to their pharmacodynamic activities. Based on LC-MS analysis of endogenous biomarkers of energy metabolism in rat plasma, it is reported that processing with ginger weakens the cold nature of Huanglian, which is consistent with the traditional processing theory that the cold nature of drugs could be moderated by processing with adjuvants of hot nature (ginger juice) [221].

Cases of hepatotoxicity due to improperly processed Radix Polygoni Multiflori (Heshouwu), the root tuber of Polygonum multiflorum Thunb. (Polygonaceae), are reported frequently [222]. Using a urinary metabolomic approach along with conventional biochemical and histopathological analysis to identify the mechanisms of hepatotoxicity, 16 potential biomarkers have been identified to be differentially expressed in rats treated with crude Heshouwu compared with those treated with processed Heshouwu. Altered vitamin B6 and tryptophan metabolism as well as a disturbed citrate cycle are found to be responsible for the hepatotoxicity of this herb. Restoring balance in these pathways is hypothesized to prevent hepatotoxicity induced by Heshouwu [223].

Using an NMR-based metabolomic approach combined with multivariate analysis by PCA, partialleast square discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA), it is revealed that the hepatoprotective effect of Chaihu is related to its effect on energy metabolism and synthesis of lipids, ketone bodies, glutathione, amino acids, and nucleotides [224]. The study identified 14 metabolites in the liver that were significantly altered after administration of Chaihu prepared by Shanxi vinegar to CCl4-treated mice in comparison with control mice. The ability of metabolomics to detect such subtle pharmacological differences sets it apart from conventional bioassays that fail to do so.

3.4. Others

Apart from color, taste and odor are also regulated as a sensory features to assess the processing quality. Intelligent sensor, a bionic technology comprises biochemical sensors and pattern recognition, has been developed to replace artificial judging which is subjective and inefficient. A taste-sensing system, named electronic tongue, was adopted to objectively evaluate the taste of processed aconite. Four types of processed aconite were unambiguously distinguished from each other using this system and each type was found to possess its own characteristic taste pattern [225]. Electrophoretic and DNA molecular marker techniques are employed to investigate the protein and DNA changes that occur during CMM processing. A two-dimensional SDS-PAGE was used to analyze the protein expression profiles of Hirudo, the dried body of Whitmania pigra Whitman (Hirudinidae), before and after stir-frying with wine. The contents of proteins in Hirudo before and after processing showed marked differences and 19 differential protein points were found [226]. DNA molecular markers are ideal genetic markers for TCM authentication. DNA fingerprinting of crude and processed Atractylodis Rhizoma (Cangzhu) was developed by randomly amplified polymorphic DNA (RAPD). The results showed DNA degradation during the processing, and the degree of degradation differed in processed products, indicating that processing conditions, such as temperature and time, greatly influence the DNA molecular identification [227].

4. Summary and future prospects

Ancient Chinese medicinal processing technology has evolved to incorporate modern processing methods and types of adjuvants to replace outdated technology, non-standard protocols, and substandard adjuvants that had become a serious impediment for the standardization and globalization of TCM. Unlike conventional processing conditions, such as heating temperature and duration, which can be controlled by automation, the sources of adjuvants used in processing demand careful selection and quality control. Therefore, research to identify the effects of adjuvant processing on the pharmacological actions of herbs holds great value. Adjuvants participate in chemical or physical transformation, directly exert pharmacological effects, or alter the pharmacokinetic behavior, to provide an enhanced therapeutic effect or counteract drug toxicity. The findings summarized in this review provide a scientific basis for the application of adjuvants and processing methods to enhance drug properties in TCM, which could bridge the gap between TCM and Western medicine.

Current research focuses on the chemical and pharmacological alterations induced by TCM processing using novel analytical methods that allow us to obtain qualitative and quantitative information of multi-component herbal products and their disposition in vivo. Synergism between drugs and adjuvants allows targeting multiple endogenous effectors, including enzymes, receptors, ion channels, transport proteins, and nucleic acids. Modern research tools, such as spectral–efficacy correlation analysis, combine analytical techniques, bioassays and stoichiometry to assign bioactivities to specific phytochemicals in herbs. TCM encompasses numerous disease symptoms that are not accurately represented by the concepts of Western medicine. Akin to the integrated concept of TCM, the introduction of systems biology allows the study of living organisms from a holistic perspective and offers an opportunity to reinvestigate TCM. With the new findings and in-depth study in this field, more and more factors have been found to influence the clinical efficacy of TCM. It is suggested that the following aspects should be taken into consideration in future studies on CMM processing.

4.1. Alteration of underlying active components in CMM processing

Current research on TCM mainly focuses on small molecules, however, macromolecules, such as polysaccharides and proteins, also exert therapeutic effects [228]. In addition, primary metabolites and inorganic ions have been overlooked, despite being vital for drug efficacy. Effects of processing methods on these active components will extend the horizons of TCM research.

4.2. Impact of processing on intestinal flora

TCMs are reported to modulate multiple pathways affecting the human gut microbial system, which contributes largely to the effectiveness of TCMs. Gut microflorae regulate the metabolism of TCMs, and also facilitate targeted physiological modulation by TCM [229]. Therefore, future studies must attempt to highlight the significance of gut microflora in drug processing.

4.3. Pharmacological and toxicological studies under pathological conditions

According to the symptom-based prescription theory, drugs that are toxic under normal physiological conditions show therapeutic effect under pathological states [230]. Pharmacological and toxicological studies have focused on drug efficacy and toxicity on healthy organisms, ignoring possible changes in drug properties induced by different pathological states. This problem is also evident in studies conducted on CMM processing. Therefore, there is a need to reappraise the therapeutic doses of drugs and redefine treatment indications after evaluating the effects of various processing methods to improve therapeutic effectiveness and avoid toxicity.