Traditional Applications,Ethnopharmacology,and Phytochemistry of Walnut Green Husk ( Juglans regia L.): A Review

Abstract

Walnut green husk (WGH) is the immature exocarp of Juglans regia L., a famous traditional herbal medicine that has long been used in China, Korea, Iran, and other places for the treatment of psoriasis, baldness, type II diabetes mellitus, intestinal worms, promotion of wound healing, and other diseases. With the advancement of modern scientific research, many studies have identified that WGH contains quinones, diarylheptane, triterpenoids, and flavonoids, and has excellent antitumor, anti-inflammatory, antibacterial, and antioxidant properties. These findings support the ethnopharmacological application of WGH and highlight the need for further investigation of the plant's potential uses in pharmaceutical fields. In this article, we made a comprehensive review of the traditional applications, botany, chemical components, pharmacological effects, and factors affecting the therapeutic properties of WGH based on a scientific literature review with a view to provide the latest information and research direction for further study and potential use of WGH. All data are gathered from scientific databases including Google Scholar, Pubmed, Sci-Finder, Web of Science, ScienceDirect, and China National Knowledge Infrastructure.

Keywords

ethnopharmacology phytochemistry quinones traditional use walnut green husk

Introduction

The walnut tree originated before Greek time and now is widely distributed throughout the world.¹ It is also called Persian walnut, White walnut, English walnut, or Common walnut.² It is native to Iran in western Asia, and was introduced into China during Zhang Qian's diplomatic mission to the Western Regions during the Han Dynasty. It can be seen that the walnut tree has a long history of cultivation in China. Walnut is a high-quality nut that contains fat, protein, sugar, dietary fiber, and vitamins.³ It is highly nutritious and has a high food value. Due to its rich nutritional value, it has been widely appreciated worldwide.⁴

In addition to this, all parts of walnut are important. As in Figure 1, walnut leaves are a rich source of natural antibacterial agents and antioxidants⁵; walnut septa have a positive effect on cough⁶; walnut brown husk have an effect of lowering blood pressure in hypertensive people⁷; walnut bark was found to have good antioxidant activity in vitro and a certain positive effect on human tumor-leukemia⁸; walnut green husk (WGH) has excellent antitumor, anti-inflammatory, antibacterial, and other pharmacological activities. However, most studies have mainly focused only on the walnut fruit (the edible part) and ignored the important role of other parts of walnut. Thankfully, with the increasing demand for natural products in nutrition, food, cosmetics, pharmaceuticals, and industry, other parts of walnut are expected to be used in a variety of industries.³

Figure 1.

The various parts of the walnut.

According to the theory of traditional Chinese medicine (TCM), WGH has the effects of pungency, bitterness, astringency, calmness, clearing away heat and detoxification, expelling wind and treating ringworm, relieving pain and stopping dysentery.⁹ WGH has long been used as a folk medicine for the treatment of various diseases, including type II diabetes mellitus (T2DM), superficial gastritis, uterine prolapse, warts, psoriasis, vitiligo, and cancer.¹⁰ Due to its long history of therapeutic use, researchers are increasingly interested in the biological activity of the herb. Recently, WGH has made gratifying achievements in phytochemistry, bioactivity, agriculture, and industry. Some recently published studies have reported various health benefits of WGH extracts, including its anti-inflammatory and antibacterial activities.¹¹ At the same time, WGH extracts have also been shown to have strong antioxidant activity,¹² and even a positive effect on breast cancer, colon cancer, ovarian cancer, lung cancer, and other cancers.¹³ All these studies provide scientific support for the use of WGH as a TCM in the treatment of these diseases.

In this article, we make a review of the phytochemistry and pharmacology of WGH by searching PubMed, Web of Science, Google Scholar, and China National Knowledge Infrastructure, and analyze the current research achievements, future research challenges and scientific gaps, hoping that this review could provide references and inspirations for future research and clinical application of WGH.

Botany

Juglandaceae, a dicotyledonous plant, is a family in the subclass Hamamelis. Walnut family plants are deciduous, semi-evergreen, or evergreen trees with pinnate compound leaves, uni-sexual flowers, double fruit skin and ovaries below.¹⁴ There are about 60 species of 8 genera in the world, mainly distributed in Asia, Europe, North America, Central America, West Indies, and South America.¹⁵ Walnut plants are suitable for growing in tropical and subtropical areas, and now there are 27 species in 7 genera in China. They are Platycarya, Engelhardia, Cyclocarya, Pterocarya, Juglans, Annamocarya, and Carya. In China, they are mostly distributed in northwest, northeast, north, and central areas such as Pinggu, Miyun, and Huairou in Beijing; Danfeng, Shangluo, and Mianxian in Shaanxi; and Yinchuan and Guyuan in Ningxia. Some species are distributed in Xinjiang and Tibet.¹⁶ A map of the distribution of walnut cultivation in China is shown in Figure 2.

Figure 2.

Map of walnut planting in China.

WGH is a dry fleshy exocarp of the walnut, which is semispherical or flaky and curled inward vertically, with a diameter of 3∼5 cm and a thickness of 6∼10 mm. The surface is smooth, looking dark green with black spots, with a broken stem at one end. The inner surface is yellowish white and uneven. WGH tastes pungent, bitter and astringent, and have the actions of clearing heat and toxins, driving away wind, treating tinea, relieving pain, and dysentery.⁹

Traditional Uses

In China, the medicinal value and traditional practices of WGH have been documented in many famous ancient classics. For example, in the 14th year of the Qianlong era, the “Fangmai Zhengzong” (The Authentic Square Vein) recorded that pounded and stir-fried WGH in an iron pot and oral administration of 15g every morning could be used to treat diarrhea. “Ben Cao Gangmu” (Compendium of Materia Medica) written by Li Shizhen of the Ming Dynasty, record application of the mixture of WGH and sulfur powder to the skin could treat vitiligo.¹⁷ In addition to the literature, WGH is also used in Chinese folklore for various diseases. Table 1 provides a list of several ancient texts and traditional uses which not only prove the long history of WGH use but provide documentary references for the modern application of WGH.

Table 1.

The Ancient Literatures and Traditional Uses of Walnut Green Husk (WGH).

	Source	Usage and curative effect	Reference
Ancient literature	Fang Mai Zheng Zong	Oral administration of 15 g WGH pounded and stir-fried in an iron pot every morning for diarrhea	17
	Manual of Shandong Chinese Herbal Medicine	Washing the affected place with hot water WGH decoction to treat acute suppurative infectious diseases	17
	Ji Jiu Fang	Washing the affected place with the mixture of mashed WGH, a small amount of soy sauce and sal ammoniac in white vinegar for various skin diseases	17
	Ben Cao Gangmu	Using the mixture of WGH and sulfur powder for vitiligo and ingrown nail	17
	Shaanxi Chinese Herbal Medicine	WGH for the treatment of psoriasis, ichthyosis, tinea lotus leaf and favus of the scalp	9
	Chinese Materia Medica	WGH for the treatment of dysentery due to damp heat, yellow and thick leucorrhea, redness and swelling of eyes, swelling of wheat grains, tears in the wind and bone tuberculosis	18
	Northeast medicine planting	WGH soaked in wine for stomach disease and abdominal pain	19
Traditional use		Oral administration of 300 ml decoction by boiling 30 g WGH in 500 ml water 30 min before meals for 30 days as a course for 2–3 consecutive courses can treat type II diabetes mellitus	20
		Oral administration of 15 ml WGH syrup 3 times daily can improve the blood status, protect the liver, and reduce inflammation. In addition, it has a good analgesic effect for the treatment of superficial gastritis and epigastric pain	10
		Oral administration of compound WGH, 6 tablets at a time 3 times daily for 2–3 courses can enhance blood circulation, promote metabolism, induce differentiation and maturation of hematopoietic cells, especially promote the recovery of stem cells and megakaryocytes suppressed due to chemotherapy, and improve the therapeutic effect and complete remission rate of leukemia	21
		WGH solution obtained by filtering crushed WGH through the gauze can be used to treat foot odor by soak the feet in warm water for 10–15 min, twice daily	22
		Scrubbing with the WGH solution obtained by filtration of decocted fresh WGH can treat ringworm and has a good effect on psoriasis and ichthyosis with continuous use	23
		WGH removes common warts by smearing fresh WGH juice over the affected area in morning and evening for about 2 weeks	24
		WGH treats uterine prolapse by promoting contraction and astringency of the uterine muscles and dispelling dampness. Contraction of the uterus is evident and itching disappears after external washing with WGH solution	25

Harvesting Period, Pretreatment, and Extraction of WGH

The period from June to September is the season for harvesting WGH. Although both immature and mature walnut pericarps can be used as medicine, this harvesting period is a bit too broad, knowing that the content of active substances in WGH may undergo changes over time. To study the dynamic accumulation rule of naphthoquinone in WGH with different harvesting time, Huo Jinhai used high performance liquid chromatography to determine the content of juglone (1) and regiolone (9), combined with the production of medicinal materials. Finally, the most suitable harvest time was determined as from mid-July to early August based on the content of total naphthoquinone (maximum content of 10.81 mg per piece), juglone and regiolone (maximum content of 6.10 mg per piece) as well as the yield of the medicinal materials.²⁶ These results are supported by Cao Guiyang et al.²⁷

Similarly, drying of medicinal herbs after extraction and different drying processes can affect the content of bioactive compounds in the herbs. Therefore, drying is a key step in preserving the bioactive components in herbs. To investigate the effect of different drying methods on the quality of WGH, 5 drying methods, including shade drying, sun drying, drying, microwave drying, and freeze drying, were evaluated by Hong Xiaoqin. The effects of different drying methods on the quality of WGH were comparatively analyzed based on the indexes of drying time, rehydration rate, water content, alcohol extract content, the content of effective components juglone (1) and regiolone (9), and the in vitro antitumor activity of WGH methanol extracts on human gastric adenocarcinoma BGC823 cells and human lung cancer A549 cells. The results showed that there was little difference in the content of alcohol extract among the 5 drying methods, and the highest content of regiolone (9) was 2.31 mg/g obtained by drying at 40°C. The growth inhibition rate of the 2 tumor cells was ordered as freeze drying > 40 °C drying > shade drying, and the inhibition rate decreased with the increase of drying temperature. Finally, from the cost, effective component content, antitumor activity and practicality comprehensive analysis, it is considered that WGH should be dried at 40 °C.²⁸

Extraction conditions and solvents also have a significant effect on the bioactive strength and chemical composition of the extract. The extraction solvent and extraction time are the key influencing factors of the extraction process, which are not only related to the cost of extraction, but need to consider the harm of organic solvents to the human body. A study reported the antimicrobial and antioxidant properties of WGH extracted from 5 different solvents (methanol, cyclohexane, ethyl acetate (EtOAc), n-butanol, and water), and the results showed that the EtOAc extract had the highest antimicrobial activity against Staphylococcus aureus SG511 compared to the other 4 extracts, with a minimal inhibit concentrations (MICs) of 3.2 mg/mL, and that its total flavonoids and polyphenols content were 42.48 mg quercetin equivalent (QE)/g and 223.25 mg of gallic acid equivalents (GAE)/g respectively. The sample extracted with EtOAc had the highest antioxidant potential (EC₅₀= 13.43 μg/mL for 1,1-diphenyl-2-picrylhydrazyl and EC₅₀= 41.83 μg/mL for 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) radical scavenging activity assay).²⁹ In addition, some scholars used the common method, reflux method, and ultrasonic method to verify the influence of different extraction methods on the extraction rate of total flavonoid and juglone (1) in WGH. The results showed that the contents of total flavonoid and juglone in the extract obtained by the ultrasonic extraction method were higher than those of the other 2 methods.³⁰

Many factors have a certain impact on the quality and activity of WGH. Although the selection of harvest time, optimization of pretreatment, screening of the extraction solvent, and extraction method increased the extraction amount of effective active substances in WGH, the preservation of WGH from the fresh product of the harvest to the drying process has not been studied. WGH is known to suffer water loss, blackening, and oxidative browning during storage. Browning reduces the content of polyphenols in WGH and decreases its antioxidant activity.³¹ Therefore, exploring the causes of WGH browning and suggesting the correct preservation method are concerns that need to be addressed in future scientific research. The research framework for WGH is listed in Figure 3.

Figure 3.

Research framework of Walnut Green Husk (WGH).

Phytochemicals Identified in WGH

WGH has yielded over 200 chemicals, including 52 quinones (1-52), 25 diarylheptanes (53-77), 51 triterpenoids and steroids (78-128), and 27 flavonoids (129-155), 35 polyphenols (156-190) and 28 other compounds (191-218). Among them, quinones are the main chemical components and main antitumor active components of WGH,⁴ diarylheptanes are the characteristic components of WGH, and polyphenols have become the main research objects at WGH in recent years. The chemical composition of WGH is introduced below, and these structures are drawn with Chemdraw (see Tables 2 to 7 and Figures 4 to 9).

Figure 4.

Part of quinones in walnut green husk.

Figure 5.

Part of diarylheptanes in walnut green husk.

Figure 6.

Part of triterpenoids and steroids in walnut green husk.

Figure 7.

Part of flavonoids in walnut green husk.

Figure 8.

Part of phenolic compounds in walnut green husk.

Figure 9.

Part of other compounds in walnut green husk.

Table 2.

the Quinone Chemistry of Walnut Green Husk (WGH).

Type	No.	Compound	Molecular formula	Extracted parts	Reference
Anthraquinones	1	Juglone	C₁₀H₆O₃	Chloroform, ethyl acetate	36
	2	2-Methoxy juglone	C₁₁H₈O₄	Chloroform	37
	3	2-Ethoxy juglone	C₁₂H₁₀O₄	Chloroform, ethyl acetate	36
	4	3-Methoxy juglone	C₁₁H₈O₄	Chloroform, ethyl acetate	36
	5	3-Ethoxy juglone	C₁₂H₁₀O₄	Chloroform, ethyl acetate	36
	6	Juglanone A	C₂₀H₁₈O₅	Ethanol	38
	7	Juglanone B	C₂₀H₁₈O₆	Ethanol	38
	8	Juglanoside B	C₁₆H₂₀O₈	Methanol	39
	9	Regiolone	C₁₀H₁₀O₃	Ethanol	40
	10	(S)-Regiolone	C₁₀H₁₀O₃	Ethanol	40
	11	Sclerone	C₁₀H₁₀O₃	Alcohol	41
	12	(4S)-45,8-Trihydroxy-α-tetralone-5-O-β- D-[6'-O-(3'‘,4'‘,5''-trihydroxybenzoyl)] glucopyranoside	C₂₃H₂₄O₁₃	n-Butyl alcohol	42
	13	1,4-Naphthol	C₁₀H₈O	n-Butyl alcohol	42
	14	1,2-Naphthoquinone	C₁₀H₆O₂	n-Butyl alcohol	42
	15	1,4-Naphthoquinone	C₁₀H₆O₂	Chloroform	37
	16	Engelharquin-one	C₂₀H₁₅O₆	Chloroform, ethyl acetate	36
	17	Xanthopurpurin	C₁₄H₈O₄	Chloroform	43
	18	2-Hydro-1,4-naphthoquinone	C₁₀H₆O₃	Ethanol	38
	19	2,5-Dihydroxy-1,4-naphthoquinone	C₁₀H₆O₄	Ethanol	38
	20	3,5-Dihydroxy-1,4-naphthoquinone	C₁₀H₆O₄	Ethanol	38
	21	5-Methoxy-1,4-naphthoquinone	C₁₁H₈O₂	Chloroform	37
	22	5,8-Dihydroxy-1-tetralone	C₁₀H₁₀O₃	Ethanol	44
	23	4-Hydroxy-1-tetralone	C₁₀H₁₀O₂	Ethanol	44
	24	4,5-Dihydroxy-α-tetralone	C₁₀H₁₀O₃	Chloroform, ethyl acetate	36
	25	(4S)-45,8-Trihydroxy-α-tetralone-4-O-β-D-glucopyranoside	C₁₆H₂₀O₉	n-Butyl alcohol	42
	26	1,8-Dihydroxy anthraquinone	C₁₄H₈O₄	Chloroform, ethyl acetate	36
	27	5,8-Dihydroxy-1,4-naphthoquinone	C₁₀H₆O₄	Chloroform	43
	28	1,3-Dihydroxy-2-methylanthraquinone	C₁₅H₁₀O₄	Chloroform	43
	29	1-Hydroxy-2-methyl-4-methoxyanthraquinone	C₁₆H₁₂O₄	Chloroform	43
	30	2,6-Dimethoxy-1,4-benzoquinone	C₈H₈O₄	Chloroform	43
	31	5,8-Dihydroxy-4-methoxy-α-tetralone	C₁₁H₁₂O₄	Ethanol	44
	32	2-Hydroxy-3-methylanthraquinone	C₁₅H₁₀O₃	Chloroform	43
	33	4,5-Dihydroxy-α-tetralone	C₁₀H₁₀O₃	Methanol	45
	34	(4S)-45,8-Thihydroxy-α-tetralone-5-O-β-D- (6'-O-4''-hydroxylbenzoyl)glucopyranoside	C₂₃H₂₄O₁₁	Chloroform	37
	35	(4S)-4,5-Dihydroxy-α-tetralone-4-O-β-D- (6'-O-4''-hydroxylbenzoyl)glucopyranoside	C₂₃H₂₄O₁₀	Chloroform	37
	36	5-Hydroxy-4-methoxy-α-tetralone	C₁₁H₁₂O₃	Ethanol	46
	37	1,7-Dihydroxy-3,6-dimethoxy-anthraquino-one	C₁₅H₁₂O₆	Ethanol	47
	38	8-Hydroxyl anthraquinone-1-carboxylic acid	C₁₀H₁₀O₃	Chloroform, ethyl acetate	48
	39	2,5-Dimethoxyp-benzoquinone	C₁₂H₁₆O₂	Ethyl acetate	49
	40	1-Hydroxy-6-methylanthraquinone	C₁₅H₁₀O₃	Ethyl acetate	49
	41	(4S)-4-Hydroxy-α-tetralone-4-O-β-D-(6'-O-4'' -hydroxylbenzoyl)glucopyranoside	C₂₃H₂₄O₉	Chloroform	37
	42	(4R)-3,4-Dihydro-4-butoxy-5-hydroxy-naphthalen-1-(2H)-one	C₁₄H₁₈O₃	Alcohol	34
	43	(4S)-4-Hydroxy-α-tetralone-4-O-β-D-glucopyranoside	C₁₆H₂₀O₇	Chloroform	37
	44	14,8-Trihydroxynaphthalene-1-O-β-D-[6'-O-(3'‘,4'‘,5'' -trihydroxybenzoyl)]glucopyranoside	C₂₃H₂₂O₁₂	n-Butyl alcohol	42
	45	14,5-Trihydroxynaphthalene-1,4-di-O-β-D-glucopyranoside	C₂₂H₂₈O₁₃	Chloroform	37
	46	14,5-Trihydroxynaphthalene-1,5-di-O-β-D-glucopyranoside	C₂₂H₂₈O₁₃	Chloroform	37
	47	(4S)-4,6-Dihydroxy-α-tetralone-4-O-β-D-glucopyranoside	C₁₆H₂₀O₈	Chloroform	37
	48	(4S)-4,5-Dihydroxy-α-tetralone-4-O-β-D-glucopyranoside	C₁₆H₂₀O₈	Chloroform	37
	49	(2R,4S,10S,12S)-2-[7-(12,13,16-trihydroxy-a-tetralonyl-13-O-b- D-glucopyranoside)]-4,8-dihydroxy-a-tetralone-4-O-b- D-glucopyranoside	C₃₂H₂₈O₁₇	Chloroform	50
	50	14,8-Trihydroxynaphthalene-1-O-β-D-glucopyranoside	C₁₆H₁₈O₈	Chloroform	37
	51	14,8-Trihydroxy-3-naphthalenecarboxylic acid -1-O-β-D-glucopyranoside ethyl ester	C₁₉H₂₂O₁₀	Chloroform	37
	52	5,8-Dihydroxy-3-methoxytetralone	C₁₁H₁₂O₄	Alcohol	51

Table 3.

The Diarylheptanoids Chemistry of Walnut Green Husk (WGH).

Type	No.	Compound	Molecular formula	Extracted parts	Reference
Diarylheptanoids	53	Juglaside A	C₂₁H₂₄O₅	Methanol	55
	54	Juglaside B	C₂₀H₂₄O₄	Ethanol	56
	55	Juglaside C	C₂₀H₂₂O₄	Ethanol	57
	56	Rhoiptelol B	C₂₀H₂₄O₆	Methanol	55
	57	Myricatomentogenin	C₂₀H₂₂O₅	Ethanol	56
	58	Engelheptanoxides A	C₂₀H₂₄O₄	Ethanol	56
	59	Myricanol	C₂₁H₂₆O₅	Alcohol	48
	60	Myricananin F	C₂₀H₂₄O₄	Ethyl acetate	39
	61	Galeon	C₂₀H₂₂O₄	Ethanol	58
	62	5-Deoxymyricanone	C₂₁H₂₄O₄	Alcohol	48
	63	(2S,3S,5S)-23,5-Trihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane	C₂₁H₂₈O₇	Methanol	55
	64	(E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-hept-1-en-3-one	C₂₁H₂₄O₅	Ethanol	56
	65	(2S,3S,5S)-2,3-Dihydroxy-5-O-β-D-xylopyranosyl-7-(4-hydroxy-3-methoxyphenyl)-1-(4-hydroxyphenyl)-heptane	C₂₅H₃₄O₁₀	Methanol	55
	66	(-)-Threo-3’,4''-epoxy-1-(4-hydroxyphenyl)-7-(3-methoxyphenyl)-heptan-2,3-diol	C₂₀H₂₄O₅	Methanol	55
	67	Tsaokoarylone	C₂₀H₂₀O₄	Methanol	55
	68	Pterocarine	C₁₉H₂₀O₄	Ethyl acetate	59
	69	1-(4'-Methoxyphenyl)-7-(3''-methoxy-2''-hydroxyphenyl)-3’,4''-epoxy-3-heptanone	C₂₁H₂₄O₅	Methanol	55
	70	3-(R)-Acetyl-1-(3’,4'-dihydroxyphenyl)-7-(4''-hydroxy-3''-methoxyphenyl)-heptane	C₂₂H₂₈O₆	Ethyl acetate	51
	71	11-Hydroxy-1,17-epoxy-7-(2-hydroxylphenyl)-13-(16-methoxyphenyl)-heptane	C₂₀H₂₄O₄	Ethyl acetate	51
	72	11-Oxo-1,17-epoxy-7-(2-hydroxylphenyl)-13-(16-methoxyphenyl)-heptane	C₂₀H₂₂O₄	Ethyl acetate	51
	73	(1α,3β,5α,6α)-1,5-Epoxy-3,6-dihydroxy-1,7-bis(3-methoxy-4-hydroxyl phenyl)-heptane	C₂₁H₂₆O₇	Ethanol	60
	74	Rhoiptelol	C₂₁H₂₆O₅	Ethanol	60
	75	5-Hydroxy-1-(4'-hydroxyphenyl)-7-(4''-hydroxy-3''-methoxy)-3-heptanone	C₂₀H₂₄O₅	Ethanol	61
	76	Hexahydrocurcumin	C₂₁H₂₆O₆	Ethanol	61
	77	1-(4'-Hydroxyphenyl)-7-(3''-methylphenyl-4''-hydroxyphenyl)-4-ene-3-heptanone	C₂₀H₂₂O₄	Ethanol	61

Table 4.

The Terpenoids Chemistry of Walnut Green Husk (WGH).

Type	No.	Compound	Molecular formula	Extracted parts	Reference
Terpenes and steroids	78	Dammara-20,24-dien-3β-ol	C₃₂H₅₂O₂	Ethanol	58
	79	2α,3β,23,24-Trihydroxy olean-12-en-28-oic acid	C₃₀H₄₈O₅	Ethanol	58
	80	Klodorol B	C₃₃H₅₀O₃	Ethanol	58
	81	1α,3β-dihydroxy-olean-18-ene	C₃₀H₄₈O₂	Ethanol	58
	82	Ursolic acid acetate	C₃₂H₅₂O₃	Ethanol	58
	83	Corosolic acid	C₃₀H₄₈O₄	Alcohol	34
	84	20(R)-24β-hydroxy-20,25-epoxy-dammar-3-one	C₃₀H₅₂O₃	Alcohol	48
	85	3β,21α-Dihydroxyurs-12-en-28-oic acid	C₃₀H₄₈O₄	Alcohol	41
	86	Pentacyclic triterpene	C₃₁H₅₀O₂	Alcohol	41
	87	2α,3β,23-Trihydroxy urs-12-en-28-oic acid	C₄₈H₇₈O₁₉	Ethanol	58
	88	Oleanolic acid	C₃₀H₄₈O₃	Ethanol	58
	89	Ursolic acid	C₃₀H₄₈O₃	Ethanol	58
	90	20(S)-Protopanaxadiol-3-one	C₃₀H₄₈O₃	Ethanol	58
	91	2α-Hydroxy oleanolic acid	C₃₀H₄₈O₄	Ethanol	63
	92	Oleanolic acid-28-O-β-D-glucopyranoside	C₃₆H₅₈O₈	Ethanol	63
	93	Arjunolic acid	C₃₀H₄₈O₅	Ethanol	63
	94	Cyclocaric acid A	C₃₀H₄₆O₃	Ethanol	63
	95	Cyclocaric acid B	C₃₀H₄₆O₅	Ethanol	63
	96	Taraxerol	C₃₀H₅₀O	Ethanol	63
	97	2α-Hydroxy ursolic acid	C₃₀H₄₈O₄	Ethanol	63
	98	23-Hydroxy ursolic acid	C₃₀H₄₈O₄	Ethanol	63
	99	Friedelin	C₃₀H₅₀O	Ethanol	63
	100	Asiatic acid	C₃₀H₄₈O₅	Methanol	39
	101	Hederagenin	C₃₀H₄₈O₄	Methanol	39
	102	Dammar-3α,12(R),20(S)-triol-12,32(R);20, 32-diepoxy-25-methy-25-entridecacyclic ether	C₃₃H₅₄O₃	Ethanol	64
	103	(23E)-12β,20(R),25(S),26-Tetrahydroxydammar-23-en-3-one	C₃₁H₅₂O₅	Ethanol	64
	104	Corosolic acid	C₂₉H₄₆O₄	Ethyl acetate	34
	105	3β,23-Dihydroxy-olean-12-en-28-oic acid	C₃₀H₄₈O₄	Ethyl acetate	34
	106	Siaresinolic acid	C₃₀H₄₈O₄	Ethanol	65
	107	Betulinic acid	C₃₀H₄₈O₃	Ethanol	65
	108	20(S)-Hydroxydammar-24-en-3-one	C₃₀H₄₉O₂	Chloroform, ethyl acetate	66
	109	20(S),24(R)-Dihydroxydammaran-25-en-3-one	C₃₀H₄₉O₃	Chloroform, ethyl acetate	66
	110	20(S),24(S)-Dihydroxydammaran-25-en-3-one	C₃₀H₄₉O₃	Chloroform, ethyl acetate	66
	111	1β,12β,20(S)-Trihydroxydammar-24-en-3-one	C₃₀H₄₈O₄	Chloroform, ethyl acetate	66
	112	20(S)-Protopanaxadiol	C₃₀H₄₉O₃	Chloroform, ethyl acetate	66
	113	3-Epikatonic acid	C₃₀H₄₉O₃	Chloroform, ethyl acetate	66
	114	3β-Hydroxyurs-20-en-28-oic acid	C₃₀H₄₉O₃	Chloroform, ethyl acetate	66
	115	2α-Hydroxy-ursolic acid	C₃₀H₄₈O₄	Chloroform, ethyl acetate	66
	116	3-oxo-23-Hydroxyurs-12-en-28-oic acid	C₃₀H₄₈O₄	Chloroform, ethyl acetate	66
	117	β-Sitosterol	C₃₀H₅₂O	n-Butanol	67
	118	β-Daucosterin	C₃₅H₆₀O₆	Methanol	39
	119	Stigmast-5-en-3β,7α-diol	C₂₉H₄₆O₂	Ethyl acetate	34
	120	Stigmast-5-en-3β,7β-diol	C₂₉H₄₆O₂	Ethyl acetate	34
	121	Stigmasterol	C₃₀H₄₈O	Ethyl acetate	34
	122	Campesterol	C₂₈H₄₈O	Ethyl acetate	34
	123	Ergosterol peroxide	C₂₈H₄₄O₃	Methanol	68
	124	Stigmast-5-en-3β-ol	C₃₁H₅₂O₂	Chloroform, ethyl acetate	43
	125	Stigmast-4-en-3-one	C₂₉H₄₈O	Chloroform, ethyl acetate	68
	126	24(R)-5α-Stigmastane-3,6-dione	C₂₉H₄₈O₂	Chloroform, ethyl acetate	68
	127	2α,3α,19α-trihydroxyurs-12-en-28-oica	C₃₀H₄₈O₄	Ethyl acetate	34
	128	3β,23-Dihydroxyurs-12-en-28-oiccaid	C₃₀H₄₈O₄	Ethyl acetate	34

Table 5.

The Flavonoids Chemistry of Walnut Green Husk (WGH).

Type	No.	Compound	Molecular formula	Extracted parts	Reference
Flavones	129	Quercetin	C₁₅H₁₀O₇	Ethanol	36
	130	Kaempferol	C₁₅H₁₀O₆	Ethanol	36
	131	(+)-Catechin	C₁₅H₁₄O₆	Ethanol	57
	132	(-)-Epicatechin	C₁₅H₁₄O₆	Ethanol	57
	133	Myricetin	C₁₅H₁₀O₈	n-Butyl Alcohol	66
	134	Crisilineol	C₁₇H₁₄O₇	Ethanol	61
	135	Sudachitin	C₁₈H₁₆O₈	Ethanol	61
	136	5,6,4'-Trihydroxy-7,3'-dimethoxy-flavone	C₁₇H₁₄O₇	Ethanol	61
	137	Eriodictyol	C₁₅H₁₀O₆	Ethanol	61
	138	Cilicione-b	C₁₅H₁₄O₈	Ethanol	61
	139	Rutin	C₂₈H₃₀O₁₄	Ethanol	61
	140	Luteolin	C₁₅H₁₀O₆	n-Butyl alcohol	66
	141	Naringenin	C₁₅H₁₂O₅	n-Butyl alcohol	66
	142	Apigenin	C₁₅H₁₀O₅	Alcohol	70
	143	Tricin	C₁₇H₁₄O₇	Alcohol	70
	144	Eupatilin	C₁₈H₁₆O₇	Alcohol	70
	145	3,5-Dihydroxy-7-methoxy-3’,4-methylenedioxyflavone	C₁₇H₁₂O₇	Alcohol	70
	146	Taxifolin	C₁₅H₁₂O₇	Alcohol	70
	147	Quercetin-3-O-(6''-galloyl)-β-D-galactopyranoside	C₂₈H₂₄O₁₆	Alcohol	70
	148	Engeletin	C₂₁H₂₂O₁₀	Alcohol	70
	149	Isoengeletin	C₂₁H₂₂O₁₀	Alcohol	70
	150	Kaempferol-3-O-β-D-glucopyranoside	C₂₁H₂₀O₁₁	Alcohol	70
	151	Quercetin-3-O-β-D-glucuronide	C₂₁H₁₈O₁₃	Alcohol	70
	152	Myricetin-3-O-β-D-glucuronide	C₂₁H₁₈O₁₄	Alcohol	70
	153	Afzelin	C₂₁H₂₀O₁₀	Alcohol	71
	154	Hyperin	C₂₁H₂₀O₁₂	Alcohol	71
	155	Pinostrobin	C₁₆H₁₄O₄	Alcohol	40

Table 6.

The Polyphenols Chemistry of Walnut Green Husk (WGH).

Type	No.	Compound	Molecular formula	Extracted parts	Reference
Phenols	156	Gallic acid	C₇H₆O₅	Petroleum ether, ethyl acetate, n-butanol	66
	157	Methyl gallate	C₈H₈O₅	Alcohol	43
	158	p-Hydroxybenzoic acid	C₇H₆O₃	Alcohol	43
	159	Methyl-3,4-dihydroxybenzoate	C₈H₈O₄	Alcohol	43
	160	6-O-Caffeic acid-D-glucose	C₁₅H₁₈O₉	Alcohol	43
	161	6-O-Gallicacid-glucoside	C₁₃H₁₆O₁₀	Alcohol	43
	162	Syringic acid	C₉H₁₀O₅	Alcohol	41
	163	Vanillic acid	C₈H₈O₄	Alcohol	41
	164	Caffeic acid	C₉H₈O₄	Alcohol	41
	165	Trans-ferulic acid	C₁₀H₁₀O₄	Alcohol	41
	166	Chlorogenic acid	C₁₆H₁₈O₉	Alcohol	41
	167	Rosmarinic acid	C₁₈H₁₆O₈	Alcohol	41
	168	1,4-Dihydroxybenzene	C₆H₆O₂	Alcohol	73
	169	Pinoresinol	C₂₀H₂₂O₆	Methanol	44
	170	2,5-Dihydroxyl methyl phenylacetate	C₉H₁₀O₄	Alcohol	73
	171	3,5-Dimethoxy-4-hydroxybenzoic acid	C₁₀H₁₀O₇	Alcohol	73
	172	p-Hydroxybenzaldehyde	C₇H₆O₂	Methanol	74
	173	Sinapic acid	C₁₁H₁₂O₅	Methanol	74
	174	Ferulic acid	C₁₀H₁₀O₄	Methanol	74
	175	p-Coumaric acid	C₉H₈O₃	Methanol	74
	176	Cilicicone b	C₁₅H₁₄O₈	Methanol	74
	177	Syringaldehyde	C₂₂H₂₆O₈	Alcohol	41
	178	Protocatechuic acid	C₇H₆O₄	Alcohol	73
	179	2-Hydroxy-4-methoxy-3,6-dimethyl benzoic acid	C₁₀H₁₂O₄	Alcohol	73
	180	Ethyl gallate	C₉H₁₀O₅	Alcohol	73
	181	Isovanillic acid	C₈H₈O₄	Methanol	44
	182	1,3-O-Dicaffeoylquinic acid	C₂₅H₂₄O₁₂	Ethyl acetate	48
	183	M-methoxyphenol	C₇H₈O₂	Ethyl acetate	48
	184	Protocatechuic acid	C₇H₆O₄	Alcohol	75
	185	Tyrosol	C₈H₁₀O₂	Alcohol	41
	186	Methyl isoferulate	C₁₁H₁₂O₄	Alcohol	75
	187	(E)-O-Hydroxycinnamic acid	C₉H₈O₃	Alcohol	76
	188	4-Hydroxypropiophenone-4-O-β-d-glucopyranosyl(1→6)-β-D-glucopyranoside	C₁₁H₃₀O₂	Alcohol	77
	189	Ellagic acid	C₁₄H₆O₈	Alcohol	76
	190	Tannic acid	C₇₆H₅₂O₄₆	Alcohol	76

Table 7.

The Other Compounds of Walnut Green Husk (WGH).

Type	No.	Compound	Molecular formula	Extracted parts	Reference
Other compounds	191	Dihydrophaseic acid	C₁₅H₂₂O₅	Alcohol	41
	192	Blumenol A	C₁₂H₂₀O₃	Alcohol	41
	193	Blumenol B	C₁₃H₂₂O₃	Alcohol	41
	194	2, 3-Dihydroxy-1-(4-hydroxy-pheny l)-propan-l-one	C₉H₁₀O₄	Alcohol	64
	195	sinapaldehyde	C₁₁H₁₂O₄	Methanol	79
	196	(Z)-10-Eicosenoic acid	C₂₀H₃₈O₂	Methanol	79
	197	(S)-3-Hydroxy-1,5-diphenyl-1-pentanone	C₁₇H₁₈O₂	Alcohol	73
	198	Dehydrovomifoliol	C₁₃H₁₈O₃	Methanol	44
	199	Loliolide	C₁₁H₁₆O₃	Methanol	44
	200	Methyl-4-hydroxy-3-methoxybenzoate	C₉H₁₀O₄	Methanol	44
	201	Succinic acid	C₄H₆O₄	Alcohol	80
	202	Methyl-4-hydroxyphenylacetate	C₉H₁₀O₃	Alcohol	47
	203	(-)-Dihydrodehy-drodiconiferyl alcohol	C₂₀H₂₄O₆	Chloroform, ethyl acetate	68
	204	Mesodihydroguaiaretic acid	C₂₀H₂₆O₄	Chloroform, ethyl acetate	68
	205	3,5-Di-O-caffeoylquinic acid butyl ester	C₂₅H₂₄O₁₂	n-Butanol	71
	206	Cinnamic acid	C₉H₈O₂	Alcohol	81
	207	Nonacosanol	C₂₉H₆₀O	Alcohol	82
	208	Hentriacontanal	C₃₁H₆₄O	Alcohol	82
	209	Hexacosanol	C₂₆H₅₄O	Alcohol	83
	210	Diisobutyl phthalate	C₁₆H₂₂O₄	Alcohol	83
	211	Dioctyl phthalate	C₂₄H₃₈O₄	Petroleum ether, ethyl acetate, n-butanol	66
	212	Hexyl-1-O-α-d-arabinofuranosyl-(1→6)-β-d-glucopyranoside	C₁₇H₃₂O₁₀	Alcohol	77
	213	Verbenol	C₁₀H₁₆O	Diethyl ether	76
	214	Verbenone	C₁₀H₁₄O	Diethyl ether	76
	215	Pinocarveol	C₁₀H₁₆O	Diethyl ether	76
	216	Pinocarvone	C₁₀H₁₄O	Diethyl ether	76
	217	Myrtenal	C₁₀H₁₄O	Diethyl ether	76
	218	Myrtenol	C₁₀H₁₄O	Diethyl ether	76

Quinone Compounds

Quinones are widely found in plants and can be classified into 4 groups: benzoquinone, naphthoquinone, phenanthrenequinone, and anthraquinone. Currently, 52 such compounds are found in WGH (1-52), of which naphthoquinones account for 70%. The abundance of quinones in WGH forms the material basis for the various biological activities of WGH. Among them, juglone (1) and regiolone (9) are of particular worthy of attention. Studies have shown that 1 and 9 have excellent biological activities such as anticancer, antioxidant, and antibacterial.³² On the one hand, it may be due to the fact that both 1 and 9 are naphthoquinone parent nuclei, which may be related to the presence of α,β-unsaturated ketone fragments in the structure, as such fragments are representative structural segments for the occurrence of Michael addition reactions, which are important for the generation of a variety of biochemical reactions. On the other hand, it is because of the high content of 1 and 9, which are easily obtained during the isolation and purification process and provide the basic conditions for the evaluation of potency and mechanism studies. They substitute different substituents to form different derivatives such as 2-methoxy juglone (2), 2-ethoxy juglone (3), 3-methoxy juglone (4), 3-ethoxy juglone (5), sclerone (11), 5,8-dihydroxy-1- tetralone (22), 4-hydroxy-1-tetralone (23) and 4,5-dihydroxy-α-tetralone (24), etc.³³

Such compounds exhibit significant biological activity, which may be related to their conformational relationship. It is worth mentioning that (4R)-3,4-dihydro-4-butoxy-5-hydroxy-naphthalen-1-(2H)-one (42) is a naphthalenone which differs from regiolone (9) in that the -OH at the R₃ position is replaced by butoxy, which has a significant cytotoxic effect, suggesting that it may have strong antitumor activity. At the same time, this important structural feature may serve as a specific chemical recognition marker in the genus walnut for distinguishing Juglandaceae from other families also rich in naphthaquinones.³⁴ Notably, a study reported the isolation of a tetraketone glycoside (2R,4S,10S,12S)-2-[7-(12,13,16-trihydroxy-a-tetralonyl-13-O-b-D-glucopyranoside)]-4,8-dihydroxy-a-tetralone-4-O-b-D-glucopyranoside (49) from WGH and compared its cytotoxic activity with that of aglycones of naphthoquinones against human cancer, showing that aglycones of naphthoquinones exhibited stronger cytotoxicity compared with tetraketone glycoside, suggesting that aglycones of naphthoquinones have a more potent antitumor effect (Table 2 and Figure 4).³⁵

Diarylheptanoids

About 25 different types of diarylheptanes were isolated from the WGH, most of which belong to the diphenyl ether structural type (53, 55, 57, 61, 66, 68, 69, 71, 72, 75, 77),^52,53 with a few of the macrocyclic biphenyl type (54, 59, 60, 62, 74) and the linear type (56, 58, 63-65 70, 76). Among them, Juglaside A (53), Juglaside B (54), and Juglaside C (55) are unique to the Juglans regional L. Cyclic diarylheptanes in the WGH often have methoxy or hydroxyl substitutions at the C-2, 3, 15, and 16 positions of the benzene ring, with fewer compounds substituted at the C-4 position. These cyclic diarylheptane compounds have various biological activities such as anti-inflammatory, free radical scavenging, antioxidant, antitumour, and immune function modulation.³⁴ It has been shown that the cyclic diarylheptane compound 3-(R)-acetyl-1-(3’,4'-dihydroxyphenyl)-7-(4''-hydroxy-3''-methoxyphenyl)-heptane (71), isolated from WGH have been shown to possess a good cholinesterase inhibitory effect, promising the development of effective drugs for the treatment of neurodegenerative diseases.²³ In addition, a small number of linear diarylheptanes in WGH have oxygen-containing substituents present at least at the C-3 position, usually ketocarbonyl or hydroxyl, and to a lesser extent acetoxy or methoxy, and these linear diarylheptanes have biological activities such as anti-inflammatory, antioxidant and insecticidal (Table 3 and Figure 5).⁵⁴

Terpenoids and Steroidals

Terpenes in WGH are mostly pentacyclic triterpenes, which account for a large proportion in WGH. Due to its structural diversity and biodiversity, extensive studies have demonstrated that they have good cytotoxic activities, such as Gerasimia Tsasi isolated the triterpene 3β,21α-dihydroxyurc-12-en-28-oic acid (85) with strong cytotoxic activity against human cancer cells.⁶²

A frequent steroidal component in WGH is β-sitosterol (117). WGH yielded 4 distinct steroids, including stigmast-5-en-3β,7α-diol (119), stigmast-5-en-3β,7β-diol (120), and β-daucosterol (118), which were all separated for the first time. The other 2 sterols detected in WGH are campesterol (122) and stigmasterol (121) (Table 4 and Figure 6).

Flavonoids

Flavonoids are not only widely distributed in the plant world but have many biological activities. Most flavonoids exist in the form of glycosides combined with sugar in plants, and some exist as a free form. At present, there are about 27 flavonoids have been isolated from WGH (129-155), of which, flavones, flavonols, dihydroflavonols, and dihydroflavonols are the main structural types.⁶⁹ In addition, 2 flavanols (131,132) and a chalcone (138) were also identified. The substituents were mainly methoxy, glucoside, and rhamnoside. Catechin (131), apigenin (142), popcornin (133), quercetin (128), kaempferol (130), and rutin (139) are the main flavonoids in WGH. Apigenin (142), a flavonoid isolated from WGH, has been shown to have remarkable cytotoxicity against Raji, MCF-7, HCT-116, and HeLa cell lines with IC₅₀ value of 5.0 ± 0.00, 5.5 ± 0.71, 12.5 ± 3.54, and 15.5 ± 4.95 μg/mL, respectively. This may be related to the -OH in 7 position of the glucuronide portion.⁶² Thankfully, apigenin only showed potent cytotoxicity to cancer cells and did not induce apoptosis in normal human cells. A series of results suggest that apigenin may prove to be a promising new anticancer drug due to its atoxic effect on normal cells (Table 5 and Figure 7).⁶²

Polyphenols

Polyphenols are compounds 156-190. The antioxidant capacity of WGH is mainly derived from phenolic compounds, showing a positive correlation between the total phenolic content and the antioxidant capacity. Macroporous resins are often used for the enrichment of polyphenols in WGH. The primary phenolic components in WGH are gallic acid (156), chlorogenic acid (166), butyric acid (162), and caffeic acid (164). Meanwhile, ellagic acid (189) and tannic acid (190) are the 2 hydrolyzed tannins present in WGH.⁷² One study confirmed that the alcoholic glycoside hexyl-1-O-d-arabinofuranosyl(16)-d-glucopyranoside (212) and the phenolic glycoside 4-hydroxypropiophenone-4-O-d-glucopyranosyl(16)-d-glucopyranoside (188) had significant inhibitory effects on tumor cells (BGC-823, HepG-2, MCF-7) and their antibacterial activity was superior to that of naphthyl glycosides such as 44, 47, and 48 (Table 6 and Figure 8).

Other Compounds

Compounds 208-213 are volatile components found in WGH, aliphatic compounds are (Z)-10-eicosenoic acid (192), nonacosanol (203), hentriacontanal (204), hexacosanol (205), and hexyl-1-O-α-darabinofuranosyl-(1→6)-β-d-glucopyranoside(212). Esters are diisobutyl phthalate (206),⁷⁶ dioctyl phthalate (207), loliolide (195), and 3,5-di-O-caffeoylquinic acid butyl ester (201).⁷⁵ It also contains potassium, calcium, zinc, copper, magnesium, iron, and other metal ions (Table 7 and Figure 9).⁷⁸

Pharmacology

Antitumor Activity

With the deepening of the research on WGH, more and more new chemical components have been found, and great progress has been made in antitumor aspects. Among the compounds isolated from WGH, juglone (1) is the most representative. Juglone has been reported to detect human intestinal tumor diversity and improve the efficacy of anticancer drugs by inhibiting the expression of the peptide prolyl isomerase Pin1.⁸⁴ Juglone has strong cytotoxic activity with an IC₅₀ value of 1.17 ± 0.3 μmol/L.³³ However, further in vivo study on its antitumor mechanism revealed that although juglone showed strong antitumor activity in vitro, it could not be used clinically as an antitumor drug due to high toxicity in vivo. Fortunately, a derivative of juglone, 3-ethyl juglone (5), can significantly reduce the toxicity to the body and maintain a high level of antitumor activity in vivo. Therefore, further research is needed to find more efficient and less toxic antitumor drugs.³⁶ Juglanin (9), another compound isolated from WGH, also has a strong tumor suppressive effect, probably by blocking the G₂/M phase of tumor cells by inducing apoptosis and autophagy of tumor cells via the reactive oxygen species/c-Jun N-terminal kinase signaling pathway with no toxic effect on normal cells. Juglanin can significantly reduce the tumor volume in vivo without causing significant weight loss.¹³ In the course of studying the inhibitory effect of compounds isolated from WGH on human cancer cells, some scholars found that oleanolic acid (88) had a strong growth inhibitory effect on human liver cancer cells HepG-2, and 2α,3β,23-trihydroxyurs-12-en-28-oic acid (87) also showed strong activity against human hepatocellular carcinoma cells (HepG-2).⁸⁵ Other studies have shown that WGH extract has a significant inhibitory effect on the growth of lung cancer cells, the mechanism of which is to downregulate cyclinD1 protein to inhibit the proliferation of lung cancer cells and upregulate tumor necrosis factor-α protein to promote the apoptosis of lung cancer cells.⁸⁶ In addition, WGH polysaccharide (WGHP) can directly inhibit or kill human colon cancer HCT-116 cell in vitro, and its antitumor mechanism may be related to inhibiting the activation of PI3 K/Akt signaling pathway.⁸⁷ All these findings demonstrate that WGH has great potential in facilitating the development of antitumor drugs. Not only the extract has a certain inhibitory effect on tumors but the compound contained in it has a strong antitumor ability. In general, it is expected to be an antitumor new drug with high medicinal value and few toxic and adverse effects.⁸⁸

Antibacterial Activity

Numerous studies have shown that WGH extracts have good antibacterial effects against most pathogenic microorganisms such as bacteria and fungi. Meihua Xi investigated and analyzed the antioxidant and antimicrobial activities of petroleum ether, EtOAc, dichloromethane, and n-butanol extracts of WGH. The results showed that the EtOAc fraction contained the highest total flavonoids (65.74 ± 1.01 mg rutin equivalents [RE]/g dry weight [DW]) and total phenols (48.73 ± 1.09 mg gallic acid equivalent [GAE]/g DW) compared to the other fractions. Moreover, the EtOAc also showed good antibacterial activity against Escherichia coli and Bacillus cereus trophoblasts with inhibitory diameters of 33.5 and 37.6 mm, respectively, and the lowest inhibitory concentration of 31.25 mg/mL. The mechanism of the antibacterial activity is that the WGH disrupts the integrity of the cell membranes, increases the permeability of the cell membranes, and triggers the leakage of intracellular substances.⁸⁹ Parvin Abedi compared the effects of WGH extract and clotrimazole on Candida albicans in female rats. The research finding that the average colony-forming units were significantly reduced after 1 week of intervention with 2% WGH extract. After 2 weeks of intervention, the average number of colony forming units in all groups was 0 except for positive control (P < .001). After 1 week of treatment with 4% WGH extract, C. albicans colony-forming unit could be eliminated, and the effect was similar to clotrimazole. The results indicated that WGH was expected to be a kind of natural antibacterial agent which could inhibit C. albicans.⁹⁰ Samaneh Dolatabadi determined the bacteriostatic effect of WGH methanolic extract using microtitre plate method. The results of the study showed that different concentrations of WGH methanolic extract had a significant effect on the growth inhibition of Pseudomonas aeruginosa (P ≤ .05), with a mean MIC value of 16 mg/mL. This suggests that WGH can be used as an alternative treatment for P. aeruginosa infection.⁹¹

Besides its use in the pharmaceutical industry, WGH can also be used in agriculture as an antibacterial agent. For example, the naphthoquinone compounds isolated from WGH can retard fungal germination. High-concentration WGH can completely inhibit the growth of Aspergillus flavus, but low-concentration WGH has a catalytic effect on aflatoxin synthesis, in which the 5-hydroxy or 2-methyl structural feature of the naphthoquinones is the key to the inhibition of aflatoxin. However, it is interesting to find that there was no significant additive effect when both substituents were present simultaneously.³⁵ For the first time, WGH was chelated with trace elements to make a fungicide against gray mold of grapes with good results. 5-Fold dilution of the WGH inhibitor was comparable to 50% carbendazim for the control of gray mold of grapes. The results showed that WGH had a good fungicidal effect and the fungicides made from WGH not only had a protective effect on the agro-ecological environment but could effectively control grape diseases, which has high economic benefits.⁹¹

Regulatory Effect of WGH on Intestinal Flora

High-fat diets can lead to obesity, intestinal microbiome imbalance and related diseases.⁹² Recent studies have shown that WGHP can reverse obesity by regulating the intestinal flora. WGHP not only reduced the abnormal weight gain, lipid metabolism disorders, inflammation, oxidative stress, and colonic tissue damage induced by high-fat diet in rats but also it improved intestinal flora dysbiosis in rats by increasing bacterial diversity in the colon and reduced the abundance of potentially pathogenic bacteria.^93,94 It is suggested that WGHP may play a protective role in high-fat diet-induced metabolic inflammation by regulating intestinal flora and short-chain fatty acids. At the same time, the protective effect of WGHP on hepatic steatosis and vascular endothelial disorder has also been confirmed.⁹⁵

In addition to WGHP, WGH extract also showed beneficial effects on intestinal flora. It was found that it could reduce weight gain and reverse hepatic total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol, malondialdehyde (MDA), alanine aminotransferase, aspartate aminotransferase and serum TC, TG, LDL-C, MDA, leptin, insulin resistance, and glucose tolerance levels in high-fat diet-induced obese rats. Thereby reducing fat accumulation, fatty liver, and adipose tissue hypertrophy. 16S rRNA sequencing demonstrated that WGH could significantly reverse HF-induced destruction of intestinal flora, reducing the relative abundance of Fusobacteria and Firmicutes at the phylum level, increasing the relative abundance of Bacteroidetes, reducing the relative abundance of Peptonococcaceae at the family level, reducing the relative abundance of Ronalbacteria and Lactobacillus, and increasing the relative abundance of Bacteroides at the genus level.⁹⁴ This result confirmed that the beneficial effects of WGH in reducing obesity and lipid disorders may be related to the regulation of intestinal flora. Qionglian Fang and Yang Yang also back up this finding.^96,97

Treat Psoriasis

WGH has long been used in traditional Chinese folklore for the treatment of psoriasis with a high cure rate.⁹⁸ However, its action mechanism has not been clearly defined. Recent studied explored the action mechanism by comparing the effects of different concentrations of WGH extract on the skin barrier function in mice with psoriasis caused by imiquimod cream. The results showed that different concentrations of WGH extract reduced the redness and swelling of the skin lesions, decreased the area of silver flakes, smoothened the skin, reduced skin thickening and relieved epidermal hyperplasia in psoriatic mice. They believed that these actions were associated with the increased expression of 4 related moisturizing factors (AQP3, Cer, Filaggrin, and Caspase14) in the skin tissue, and the therapeutic effect was linearly correlated with the dosage. These findings provide experimental and documentary support for the ancient documentation and folk applications of WGH for the treatment of psoriasis.⁹⁹

Promote Wound Healing

In Iranian traditional medicine, WGH is considered as a good remedy for would healing. This wound-healing effect of WGH has also been confirmed by experimental studied. The researchers divided 48 male Wistar albino rats into 4 groups of 12 rats each. An incision was made at the back of each rat. WGH extract (20% w/w), WGH burnt residues (20% w/w), Eucerin, and Phenytoin ointments were used in each group. Wound length, shrinkage, and histopathological evaluations were recorded on days 3, 7, 10, and 14, respectively. The result showed that the physiopathological indexes were improved significantly in rats of the WGH extract group as compared with the control group (P ≤ .05), and the effect of WGH extract was better than that of WGH slag, demonstrating that WGH has a promising application potential in promoting wound healing and suggesting that WGH may be a valuable resource for developing new trauma healing drugs.¹⁰⁰

Analgesic Activity

According to the Compendium of Materia Medica written by Li Shizhen more than 1200 years ago, WGH had the effect of “relieving heavy pain in the low back and legs” and “relieving pains in the heart and abdomen.” In recent years, some scholars have demonstrated through experiments that potassium salt is the main analgesic component in WGH.¹⁹ They found that that pain could be relieved within 10 min after WGH application as an analgesic in more than subjects, and believed that this analgesic effect was attributed to the potassium salt in WGH under the action of the central nervous system by reducing the Ca²⁺ content and increasing the K⁺ content in the brain. At the same time, it stimulated certain neurons to release neurotransmitters to modulate the nociceptive response. These experimental results are consistent with the clinical observations, indicating that there is great potential for the clinical application of WGH as an analgesic.¹⁰¹

Other Pharmacological Activities

Some studies observed the effect of WGH Astragalus Root decoction by WGH, astragalus root, rhizoma dioscoreae, radix trichosanthis, radix rehmanniae, lycium chinense miller, rhizoma coptidis, salvia miltiorrhiza for the treatment of T₂DM and found that it had a good effect.¹⁰² Also, WGH has good antibacterial and analgesic effects against gastric and duodenal ulcer, pain from gastritis, ringworm sores, and favus of the scalp. The pharmacological effects and mechanism of WGH are listed in Table 8.

Table 8.

Pharmacological Effects of Walnut Green Husk (WGH).

Therapeutic benefits	Efficacy of parts	Mechanism	Results	Reference
Breast cancer	Juglanin	WGH induces G₂/M phase block in human breast cancer cells and induces apoptosis and autophagy through the reactive oxygen species/c-Jun N-terminal kinase signaling pathway. It induces apoptosis in breast cancer cells by downregulating Bcl-2 and upregulating Bax, Caspase-9 and Caspase-3 reversing protein expression	WGH induces apoptosis and autophagy in breast cancer cells	¹³
Leukemia	WGH extract	WGH extract induces early apoptosis and loss of mitochondrial membrane potential	WGH extract has an antiproliferation effection HL-60 cells	⁸⁸
Gastric cancer	WGH extract;3-Ethyl juglone	WGH inhibits tumor cell growth by upregulating Bax expression and downregulating Bcl-2 expression. It promotes tumor cell apoptosis through downregulation of vascular endothelial growth factor protein expression	WGH has a concentration-dependent inhibitory effect on the proliferation of gastric cancer cells SGC-7901	⁴⁵
Prostate cancer	WGH cyclohexane extract	The mechanism by which WGH inhibits prostate cancer through upregulation of Bax, downregulation of Bcl-2 and activation of caspases to induce apoptosis in prostate cancer cells	WGH cyclohexane extract can significantly inhibit the growth of prostate cancer cells	¹⁰³
Esophageal cancer	WGH extract	WGH blocks the KYSE150 cycle in esophageal cancer cells in the G₀/G₁ phase and induces apoptosis via the mitochondrial pathway	WGH extract has significant cytotoxicity to esophageal carcinoma cells	¹⁰⁴
Lung cancer	WGH extract	WGH crude extract inhibited lung cancer cell proliferation by downregulating cyclin D₁ protein and promoted lung cancer cell death by upregulating tumor necrosis factor-α protein	WGH crude extract has certain lethality to lung cancer cells	⁸⁶
Intestinal tumor	Juglone	Juglone inhibits the overexpression of the peptideproline isomerase Pin1 and improves the efficacy of anticancer drugs	WGH improves the efficacy of anticancer drugs	³³
Colon cancer	WGH polysaccharide	Its antitumor mechanism is related to inhibiting the activation of PI3 K/Akt signaling pathway	WGH polysaccharide can directly inhibit or kill human colon cancer HCT-116 cell in vitro	⁸⁷
Pseudomonas aeruginosa	Juglans rega L. extract	WGH kills bacteria by destroying the biofilm on the body surface	WGH has a significant influence on the growth and biofilm inhibition of pseudomonas aeruginosa	⁹¹
Aflatoxin	Naphthoquinoner isolated from WGH	The structural characteristics of the 5-hydroxy or 2-methyl naphthoquinones are key to the inhibition of aflatoxins	The naphthoquinones in WGH completely inhibit the growth of Aspergillus flavus at high concentrations, but promote the synthesis of A. flavus at low concentrations	³⁵
Gray mold of grape	WGH was chelated with trace elements to make a fungicide	-	The control effect of WGH inhibitor 5 times dilution on grapevine gray mold was comparable to that of 50% carbendazim	¹⁰⁵
Regulate intestinal flora	WGHP	WGHP may play a protective role in metabolic inflammation induced by high fat diet by regulating intestinal flora and short-chain fatty acids	WGH improves intestinal flora	⁹⁵
Reduce fat accumulation, fatty liver, and fatty tissue hypertrophy	WGH extract	The beneficial effects of WGH extract on reducing obesity and lipid disorders related to the regulation of intestinal flora	WGH improves intestinal flora	⁹³
Psoriasis	WGH extract	WGH improves the redness of skin lesions by increasing the expression of 4 related moisturizing factors, AQP₃, Cer, Filaggrin, and Caspase14, in skin tissues, and the therapeutic effect is linearly related to the dose	Different concentrations of WGH extract reduced the redness and swelling of skin lesions, decreased the area of silver flakes, smoothened the skin, reduced skin thickening, and relieved epidermal hyperplasia in psoriatic mice	⁹⁹
Wound healing	WGH extract	-	WGH can promote wound healing in rats	¹⁰⁰
Analgesic	WGH extract	WGH can reduce Ca²⁺ content and increase K⁺ content in brain, and stimulate some neurons to release neurotransmitters and regulate pain response	Pain relief within 10 min in over 90% of people when WGH is applied to analgesia	¹⁰¹

Conclusion

WGH is a famous traditional herb widely used in China, North Korea, Iran, and other places. At present, 218 compounds have been isolated and identified from WGH. There were 52 quinones, 25 diarylheptane compounds, 51 triterpenoids and steroids, 27 flavonoids, 35 polyphenols and 28 other compounds. Among them, quinone compounds are the main chemical components of WGH, and also the potential pharmacodynamic material basis. With the development of modern pharmacology, the research of WGH has been carried out from the aspects of antitumor, anti-inflammatory, and antibacterial. Unlike previously, in recent years, pharmacological studies on WGH have gradually leaned toward traditional folk applications such as psoriasis, analgesia, and regulation of intestinal flora, rather than being limited to modern pharmacological applications such as antitumor, as shown in Figure 10. Although there are few reports on the traditional treatment effect of WGH at present, we can see the trend of basic research toward traditional application, which is necessary to further characterize the relationship between traditional application of WGH and modern pharmacology.

Figure 10.

Number of literatures on pharmacological effects of walnut green husk before and after 2015.

Footnotes

Author Contribution Statement

BL collated documents, wrote, and finalized the manuscript; CC reviewed the literature, discussed the layout, finished the artworks (figures and tables), and finalized the manuscript; CZ directed the pharmacology section; FH retrieved the relevant literature and discussed the layout. LH and CB designed the manuscript.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Key Research & Development Program of Ningxia (grant number No. 2021BEG03102).

ORCID iDs

Changcai Bai

Shijie Wei

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