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Abstract With quercetin content and yield of dry extract as investigation indices， three factors， amount of water added， decoction time and decoction times were investigated by an orthogonal test， so as to select the optimal water extraction process for Zihua Qinghuo Capsule. The determined extraction process was adding 8 times of water in five herbs including Viola philippica and decocting for 2 times， 1.5 h each time. Pilot scale test demonstrated that the selected process is stable and feasible， and could provide reliable reference for practical production.
Key words Zihua Qinghuo Capsule； Extraction process； Quercetin； Orthogonal test
Zihua Qinghuo Capsule is a pure Chinese herbal medicine compound preparation developed by the second Affiliated Hospital of Guiyang College of Traditional Chinese Medicine from five kinds of Chinese herbs including Viola philippica and Scrophularia ningpoensis Hemsl. It has the effects of clearing away heat and toxic materials， treating yin deficiency by reinforcing body fluid and strengthening teeth and gingiva， and is used for treating gingival bleeding， toothache and peridental abscess caused by chronic marginal gingivitis and adult periodontitis[1]. The preparation is prepared by decocting the five Chinese herbs including V. philippica and S. ningpoensis in water together. The main herbs in the prescription， V. philippica and Taraxacum mongolicum contain many effective components including flavonoids， coumarins and polysaccharides， which have the pharmacologic action of resisting inflammation， inhibiting bacteria， resisting viruses， resisting oxidation and promoting wound healing[2-3]， which accords with the efficacy of the prescription. In order to better improve the quality of the preparation and ensure its curative effect， with quercetin content and yield of dry extract after hydrolysis of flavonoids as investigation indices， an orthogonal test was carried out to optimize the water extraction conditions for Zihua Qinghuo Capsule.
Instruments and Reagents
The instruments included an LC??2010AT high performance liquid chromatograph （Shimadzu） equipped with a UV detector and Class??VP6.12 chromatographic work station， and an FA1004 electronic balance （Shanghai Precision Instruments Co.， Ltd.）. The used reagents mainly included quercetin reference substance （lot number： 11182??201212， National Institutes for Food and Drug Control）， chromatographically pure methanol and redistilled water， and other reagents were all analytically pure. Chromatographic Conditions
HPLC was performed using a Diamonsil C18 column （250 mm??4.6 mm， 5 ??m） with methanol -0.4% phosphoric acid solution （50?? 50） as the mobile phase at a flow rate of 1.0 ml/min and column temperature of 5 ??. Samples were injected at an amount of 10 ??l and detected at 360 nm. The number of theoretical plates was calculated according to quercetin peak to be not lower than 3 000.
Methods and Results
Extraction factors of orthogonal test
With decoction pieces of five Chinese herbs including V. philippica and S. ningpoensis as test materials， three factors， amount of water added， decoction time and decoction times were investigated， and each factor was selected with three different levels. Yield of dry extract and extracting amount of quercetin were selected as the optimum??seeking indices to determine a rational process. Under a parallel operation condition， an orthogonal test was designed according to L9 （34） table. The levels of factors are shown in Table 1.
Preparation of sample solution and calculation of yield of dry extract
Herbs including T. mongolicum in the prescription were mixed proportionally （1/10） and divided into equal parts， each of which had a weight of 165 g. The samples were extracted according to the design of the orthogonal test， and filtered， giving filtrates， which were concentrated into thick pastes. The thick pastes were then dried at 80 ?? to a constant weight and cooled in a dryer for 0.5 h， followed by weighing and calculation of yield of dry extract.
Determination of quercetin content in dry extract
About 1.0 g of the sample powder was accurately weighed and added into a conical flask. Then， 50 ml of methanol??hydrochloric acid （4?? 1） solution was accurately added into the conical flask and sealed with a cover， followed by weighing. The solution was refluxed for 1 h with heating， followed by cooling and weighing. Methanol was added to complement the lost weight， and the solution was shaken and filtered， obtaining the filtrate for later determination.
A proper amount of quercetin reference substance dried to constant weight were accurately weighed and added with methanol， obtaining a 30 ??g/ml reference solution.
Finally， 10 ??l of each of the reference solution and test solution was accurately measured， injected to HPLC and determined.
Scheme and results of the orthogonal test and variance analysis
It could be seen from Table 3 and Table 4 that with yield of dry extract as an evaluation index， factor A had a significant difference， while factors B and C had no significant differences. Comprehensively from above analysis， A1B1C1 was optimal. With extracting amount of quercetin as an evaluation index， factors A， B and C all had significant differences. For factor A， the three levels ranked as A3>A2>A1； in the case of factor B， the order was B3>B2>B1； and as to factor C， its three levels were in order of C2>C3>C1. Because extracting amount of quercetin is more important than yield of dry extract， A3， B3 and C2 were selected， i.e.， A3B3C2 was optimal. It was thus determined that A3B3C2 was the optimal process. Discussion
It could be know by consulting literatures that the water extract of this prescription contains the effective components， flavonoids， which produce quercetin during hydrolysis. Quercetin serving as an index could represent one of the main medicinal components in the prescription， and combining with yield of dry extract， the optimal water extraction process for Zihua Qinghuo Capsule could be selected.
With reference to the determination method of quercetin content under the item of Polygonum perfoliatum L. in Pharmacopeia of China （2015）[4]， an HPLC method for determining quercetin in Zihua Qinghuo Capsule was established by methodological investigation. During the investigation， a negative sample solution was prepared from a double??negative sample lacking T. mongolicum and V. mandshurica according to the method for preparing test solution. We also investigated the extraction method. Ultrasonic and refluxing methods were compared， and the reflux extraction method was selected with an extraction time of 1 h.
In this study， the extraction process of Zihua Qinghuo Capsule prescription was optimized， and the determined extraction process was adding 8 times of water in five herbs including V. mandshurica and decocting for 2 times， 1.5 h each time.
References
[1] ZHAO H， ZHOU Q， CHEN MQ. Preparation and preliminary clinical observation of Zipu Capsule[J]. Agricultural Science & Technology， 2016， 17（7）： 1733-1735， 1743.
[2] WU Q， GAO YP. Research advances on chemical constituents and pharmacological actions of Viola yedoensis[J]. Chinese Journal of Ethnomedicine and Ethnopharmacy， 2017， 22： 38-38.
[3] WANG QY. Advances in extraction and application of effective components in T. mongolicum[J]. Jiangsu Agricultural Sciences， 2016， 8： 21-24.
[4] Chinese Pharmacopoeia Commission. Pharmacopeia of China[S]. Beijing： China Medical Science and Technology Press， 2015： 166-167.
Key words Zihua Qinghuo Capsule； Extraction process； Quercetin； Orthogonal test
Zihua Qinghuo Capsule is a pure Chinese herbal medicine compound preparation developed by the second Affiliated Hospital of Guiyang College of Traditional Chinese Medicine from five kinds of Chinese herbs including Viola philippica and Scrophularia ningpoensis Hemsl. It has the effects of clearing away heat and toxic materials， treating yin deficiency by reinforcing body fluid and strengthening teeth and gingiva， and is used for treating gingival bleeding， toothache and peridental abscess caused by chronic marginal gingivitis and adult periodontitis[1]. The preparation is prepared by decocting the five Chinese herbs including V. philippica and S. ningpoensis in water together. The main herbs in the prescription， V. philippica and Taraxacum mongolicum contain many effective components including flavonoids， coumarins and polysaccharides， which have the pharmacologic action of resisting inflammation， inhibiting bacteria， resisting viruses， resisting oxidation and promoting wound healing[2-3]， which accords with the efficacy of the prescription. In order to better improve the quality of the preparation and ensure its curative effect， with quercetin content and yield of dry extract after hydrolysis of flavonoids as investigation indices， an orthogonal test was carried out to optimize the water extraction conditions for Zihua Qinghuo Capsule.
Instruments and Reagents
The instruments included an LC??2010AT high performance liquid chromatograph （Shimadzu） equipped with a UV detector and Class??VP6.12 chromatographic work station， and an FA1004 electronic balance （Shanghai Precision Instruments Co.， Ltd.）. The used reagents mainly included quercetin reference substance （lot number： 11182??201212， National Institutes for Food and Drug Control）， chromatographically pure methanol and redistilled water， and other reagents were all analytically pure. Chromatographic Conditions
HPLC was performed using a Diamonsil C18 column （250 mm??4.6 mm， 5 ??m） with methanol -0.4% phosphoric acid solution （50?? 50） as the mobile phase at a flow rate of 1.0 ml/min and column temperature of 5 ??. Samples were injected at an amount of 10 ??l and detected at 360 nm. The number of theoretical plates was calculated according to quercetin peak to be not lower than 3 000.
Methods and Results
Extraction factors of orthogonal test
With decoction pieces of five Chinese herbs including V. philippica and S. ningpoensis as test materials， three factors， amount of water added， decoction time and decoction times were investigated， and each factor was selected with three different levels. Yield of dry extract and extracting amount of quercetin were selected as the optimum??seeking indices to determine a rational process. Under a parallel operation condition， an orthogonal test was designed according to L9 （34） table. The levels of factors are shown in Table 1.
Preparation of sample solution and calculation of yield of dry extract
Herbs including T. mongolicum in the prescription were mixed proportionally （1/10） and divided into equal parts， each of which had a weight of 165 g. The samples were extracted according to the design of the orthogonal test， and filtered， giving filtrates， which were concentrated into thick pastes. The thick pastes were then dried at 80 ?? to a constant weight and cooled in a dryer for 0.5 h， followed by weighing and calculation of yield of dry extract.
Determination of quercetin content in dry extract
About 1.0 g of the sample powder was accurately weighed and added into a conical flask. Then， 50 ml of methanol??hydrochloric acid （4?? 1） solution was accurately added into the conical flask and sealed with a cover， followed by weighing. The solution was refluxed for 1 h with heating， followed by cooling and weighing. Methanol was added to complement the lost weight， and the solution was shaken and filtered， obtaining the filtrate for later determination.
A proper amount of quercetin reference substance dried to constant weight were accurately weighed and added with methanol， obtaining a 30 ??g/ml reference solution.
Finally， 10 ??l of each of the reference solution and test solution was accurately measured， injected to HPLC and determined.
Scheme and results of the orthogonal test and variance analysis
It could be seen from Table 3 and Table 4 that with yield of dry extract as an evaluation index， factor A had a significant difference， while factors B and C had no significant differences. Comprehensively from above analysis， A1B1C1 was optimal. With extracting amount of quercetin as an evaluation index， factors A， B and C all had significant differences. For factor A， the three levels ranked as A3>A2>A1； in the case of factor B， the order was B3>B2>B1； and as to factor C， its three levels were in order of C2>C3>C1. Because extracting amount of quercetin is more important than yield of dry extract， A3， B3 and C2 were selected， i.e.， A3B3C2 was optimal. It was thus determined that A3B3C2 was the optimal process. Discussion
It could be know by consulting literatures that the water extract of this prescription contains the effective components， flavonoids， which produce quercetin during hydrolysis. Quercetin serving as an index could represent one of the main medicinal components in the prescription， and combining with yield of dry extract， the optimal water extraction process for Zihua Qinghuo Capsule could be selected.
With reference to the determination method of quercetin content under the item of Polygonum perfoliatum L. in Pharmacopeia of China （2015）[4]， an HPLC method for determining quercetin in Zihua Qinghuo Capsule was established by methodological investigation. During the investigation， a negative sample solution was prepared from a double??negative sample lacking T. mongolicum and V. mandshurica according to the method for preparing test solution. We also investigated the extraction method. Ultrasonic and refluxing methods were compared， and the reflux extraction method was selected with an extraction time of 1 h.
In this study， the extraction process of Zihua Qinghuo Capsule prescription was optimized， and the determined extraction process was adding 8 times of water in five herbs including V. mandshurica and decocting for 2 times， 1.5 h each time.
References
[1] ZHAO H， ZHOU Q， CHEN MQ. Preparation and preliminary clinical observation of Zipu Capsule[J]. Agricultural Science & Technology， 2016， 17（7）： 1733-1735， 1743.
[2] WU Q， GAO YP. Research advances on chemical constituents and pharmacological actions of Viola yedoensis[J]. Chinese Journal of Ethnomedicine and Ethnopharmacy， 2017， 22： 38-38.
[3] WANG QY. Advances in extraction and application of effective components in T. mongolicum[J]. Jiangsu Agricultural Sciences， 2016， 8： 21-24.
[4] Chinese Pharmacopoeia Commission. Pharmacopeia of China[S]. Beijing： China Medical Science and Technology Press， 2015： 166-167.