Baseline data collections of lipopolysaccharide content in 414 herbal extracts and its role in innate immune activation

Abstract

Some herbal extracts contain relatively high amounts of lipopolysaccharide (LPS). Because orally administered LPS activates innate immunity without inducing inflammation, it plays a role as an active ingredient in herbal extracts. However, the LPS content in herbal extracts remains extensively unevaluated. This study aimed to create a database of LPS content in herbal extracts; therefore, the LPS content of 414 herbal extracts was measured and the macrophage activation potential was evaluated. The LPS content of these hot water extracts was determined using the kinetic–turbidimetric method. The LPS concentration ranged from a few ng/g to hundreds of μg/g (Standard Escherichia coli LPS equivalent). Twelve samples had a high-LPS-content of > 100 μg/g, including seven samples from roots and three samples from leaves of the herbal extracts. These samples showed high phagocytosis and NO production capacity, and further investigation using polymyxin B, an LPS inhibitor, significantly inhibited macrophage activation. This study suggests that some herbal extracts contain sufficient LPS concentration to activate innate immunity. Therefore, a new approach to evaluate the efficacy of herbal extracts based on their LPS content was proposed. A database listing the LPS content of different herbal extracts is essential for this approach.

Introduction

Lipopolysaccharide (LPS) is a lipid and polysaccharide molecule found in the outer membrane of gram-negative bacteria^1,2. LPS has long been considered an endotoxin owing to its wide use as a potent inflammation inducer because it binds to Toll-like receptor (TLR4)^3–6 of immune cells and activates nuclear factor-kappa beta (NFκB)^7–9 to cause inflammatory cytokines, including interleukin-1 beta (IL-1β)^10–12, interleukin-6 (IL-6)¹³, and tumor necrosis factor alpha (TNFα)^14,15, inducing severe fever, diarrhea, and shock when intravenously injected^16–20. Furthermore, although oral administration of LPS does not induce inflammation in healthy subjects, it has been observed that disrupted barrier system and bacterial translation may occur in diseases with persistent inflammatory lesions in the intestinal tract and periodontal tissues. Experimental models in which persistent bacterial and LPS invasion in vivo induces systemic inflammation suggest the involvement of LPS in chronic inflammatory diseases, including lifestyle-related diseases²¹.

However, gram-negative bacteria with LPS are found in large amounts in the human intestinal tract²², skin^23,24, and other organs in contact with the outside world without causing any inflammatory effects under healthy conditions²⁵. The decreased number of these gram-negative bacteria in the intestinal tract resulting from the use of antibiotics causes a decrease in the amount of antimicrobial peptides 59^26,27, making individuals more susceptible to infections^28,29. Thus, LPS in the intestinal tract and skin has been suggested to play a beneficial role in maintaining health. Furthermore, the lack of exposure to LPS is associated with susceptibility to allergic and infectious diseases^30,31. This shows that LPS have unknowingly been taken orally and transdermally to maintain our health.

In a previous study, it was revealed that LPS is present in many plants, including herbal extracts³². It also known that several LPSs are present in rice and wheat, which are staple foods, and that their ingestion confers functional properties. Additionally, Pantoea agglomerans was isolated as the dominant LPS symbiont in wheat³³. Oral consumption of Pantoea agglomerans LPS (LPSp) enhanced phagocytosis of abdominal macrophages in mice, but this effect was not observed in TLR4-deficient mice³⁴. This indicates that orally administered LPS promotes foreign body removal via innate immunity using TLR4. Furthermore, in disease prevention and treatment experiments, oral LPSp administration was found to enhance the effect of anticancer drugs³⁵, promote the treatment of lung metastases³⁶, inhibit itching in atopic dermatitis²⁵, prevent atherosclerosis in apolipoprotein-E (ApoE)-deficient mice³⁷, prevent dementia in brain diabetes-induced mice³⁸ etc. Additionally, a recent study reported that orally administered LPS suppressed diabetic symptoms by increasing the expression of insulin signaling-related factors, especially adiponectin, in adipose tissue in type 2 diabetes mellitus, a disease supposedly LPS-induced³⁹. Furthermore, LPSp has been confirmed to be highly safe in rats, with no adverse effects after oral administration at 2 g/kg body weight (BW) or higher⁴⁰.

From the above-mentioned studies, LPS from ingested food is likely to activate and regulate innate immunity. Furthermore, considering its presence in herbal extracts, there is a possibility that the consumption of herbal extracts may activate the body innate immunity regulation. Herbal extracts are defined as naturally occurring unrefined substances from any part or parts of plants, animals, and other organisms with one or more active ingredients intended to alleviate, treat, or prevent diseases⁴¹. The above-mentioned wheat is a herbal extract listed in the “The Japanese standards for nonpharmacopoeial herbal extracts 2022” and is called Shobaku⁴². The overall health benefits of consuming herbal extracts are generally thought to be due to the low molecular weight of the active ingredient. However, a sufficient amount of LPS in the herbal extracts can activate the innate immune system; therefore, LPS should also be considered an active ingredient of herbal extracts. As the innate immune system-activating effect of orally administered LPS is coming to light³⁴, LPS in herbal extracts as a component of the effects of Chinese herbal medicine deserves attention. Thus, a database of the LPS content in herbal extracts and food ingredients is required to make this concept common knowledge.

In 1992, our group screened approximately 60 plant samples, including herbal extracts, for their LPS content and found that some plants had a high LPS content of over 100 μg/g³². However, since then, little effort has been made to measure the LPS content in herbal extracts. Montenegro et al. was the first to report on LPS’s ability to activate macrophages, an innate immunity mechanism, in Kampo medicine⁴³. In this study, they showed that the macrophage-activating component of Juzen-taiho-to, an immune-boosting Kampo medicine formulated from 10 herbal extracts, is correlated with the amount of LPS, which is obtained from symbiotic bacteria existing in one of its ingredients. Their study showed that LPS is a functional component that activates and controls macrophages (innate immunity) in Juzen-taiho-to; hence, LPS can be regarded as an active component of the innate immune system of numerous herbal extracts because most herbal extracts have symbiotic bacteria that supply LPS. Therefore, if information on the LPS content found in herbal extracts can be obtained, the knowledge that oral intake of LPS does not induce inflammation can be enforced, and a new perspective on the concept of LPS as an effective component of herbal extracts can be provided. However, data evaluating herbal extracts from the LPS viewpoint are currently extremely limited, as described above.

Thus, to provide a comprehensive list of the LPS content of herbal extracts and other food ingredients, the LPS content of 414 herbal extracts were measured and compared. Additionally, the macrophage activation potential of herbal extracts with particularly high-LPS-content was compared and measured to investigate the connection between LPS content and macrophage activity.

Results

Measurement of the LPS content of herbal extracts

By measuring Limulus activity, the amount of LPS in the herbal extracts was examined. The LPS concentrations of 414 samples of herbal extracts obtained from vascular plants, fungi, and others ranging from below the detection limit to several 100 μg/g are shown in Table 1. Figure 1 shows the distributions of the LPS concentrations within each species. Herbal extracts from vascular plants were further divided according to their parts. For this analysis, the groups were classified according to the crude drug classification method. The results showed that herbal extract ingredients with high LPS contents were mostly found in the vascular plant group. Comparisons between vascular plant parts indicated that roots (107 samples) had significantly higher LPS levels than fruits (69 samples) and seeds (22 samples), and leaves (68 samples) had significantly higher LPS levels than fruits (69 samples). The average LPS concentration in all samples was 17.4 ± 69.3 μg/g. There are 12 samples containing high LPS concentration > 100 μg/g, 80 samples containing concentrations of 10–100 μg/g, and 162 samples containing concentrations of 1–10 ng/g. The 12 samples with significantly high LPS contents, which are listed in Table 2, were selected to further test the macrophage-activating effect of LPS. The measured LPS content indicated that herbal extracts contain LPS and that the amount of LPS in each plant’s part varies depending on the parts from which they are derived.

Table 1.

LPS concentrations of 414 samples of herbal extracts. For herbal extracts with multiple scientific names, the scientific names listed in this table are those most used in Japan.

No	Species	English name	Scientific name	Part	Limulus activity (μg/g)
1	Plant	Achyranthes root	Achyranthes bidentata Blume	Root	0.391
2	Plant	Aconite root	Aconitum carmichaelii Debeaux	Root (Tuberous root)	2.705
3	Plant	Actinidia gall	Actinidia polygama (Siebold & Zucc.) Planch. ex Maxim	Fruit (Gall)	3.891
4	Plant	Adenophora root	Adenophora triphylla (Thunb.) A.DC	Root	5.984
5	Plant	Agarwood	Aquilaria malaccensis Lam	Stem (Xylem)	16.284
6	Plant	Agrimony	Agrimonia eupatoria L.	Stem and Leaf	3.282
7	Plant	Ajuga herb	Ajuga decumbens Thunb	Whole plant	10.642
8	Plant	Akebia fruit	Akebia quinata (Thunb. ex Houtt.) Decne	Fruit	12.147
9	Plant	Akebia stem	Akebia quinata (Thunb. ex Houtt.) Decne	Stem	1.962
10	Plant	Alfalfa	Medicago sativa L.	Stem and Leaf	4.089
11	Plant	Alisma tuber	Alisma plantago-aquatica subsp. orientale (Sam.) Sam	Root (Rhizome)	0.409
12	Plant	Allium chinense bulb	Allium chinense G.Don	Root (Bulb)	6.554
13	Plant	Allspice	Pimenta dioica (L.) Merr	Fruit	3.543
14	Plant	Amomum seed	Wurfbainia villosa var. xanthioides (Wall. ex Baker) Škorničk. & A.D.Poulsen	Seed	6.392
15	Plant	Amomum tsao-ko fruit	Lanxangia tsao-ko (Crevost & Lemarié) M.F.Newman & Škorničk	Fruit (Mature fruit)	0.018
16	Plant	Anemarrhena rhizome	Anemarrhena asphodeloides Bunge	Root (Rhizome)	38.905
17	Plant	Angelica	Angelica archangelica L.	Root	29.527
18	Plant	Angelica dahurica root	Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav	Root	266.554
19	Plant	Anise	Pimpinella anisum L.	Fruit	0.100
20	Plant	Apple	Malus domestica (Suckow) Borkh	Fruit	8.276
21	Plant	Apricot kernel	Prunus armeniaca L.	Seed	0.009
22	Plant	Aralia rhizome	Aralia cordata Thunb	Root (Rhizome)	503.986
23	Plant	Aralia root	Aralia cordata Thunb	Root	31.014
24	Plant	Aralia elata root bark	Aralia elata (Miq.) Seem	Root (Root bark)	3.891
25	Plant	Areca	Areca catechu L.	Seed	0.808
26	Plant	Arisaema tuber	Arisaema heterophyllum Blume	Root (Tuber)	1.488
27	Plant	Arnica flower	Arnica montana L.	Flower	29.527
28	Plant	Artemisia leaf	Artemisia princeps Pamp	Leaf	121.750
29	Plant	Artichoke	Cynara cardunculus L.	Stem and Leaf	4.642
30	Plant	Ash bark	Fraxinus chinensis subsp. rhynchophylla (Hance) A.E.Murray	Stem (Bark)	1.251
31	Plant	Ashitaba	Angelica keiskei (Miq.) Koidz	Leaf	0.220
32	Plant	Asparagus	Asparagus officinalis L	Stem	0.066
33	Plant	Asparagus root	Asparagus cochinchinensis (Lour.) Merr	Root	2.389
34	Plant	Aster root	Aster tataricus L.f.	Root (Root and Rhizome)	13.635
35	Plant	Astragalus root	Astragalus mongholicus Bunge	Root	1.256
36	Plant	Asunaro	Thujopsis dolabrata (L.f.) Siebold & Zucc	Branch and Leaf	1.445
37	Plant	Atractylodes lancea rhizome	Atractylodes lancea (Thunb.) DC	Root (Rhizome)	5.609
38	Plant	Bamboo culm	Bambusa textilis McClure	Stem (Culm)	1.151
39	Plant	Banaba	Lagerstroemia speciosa (L.) Pers	Leaf	0.363
40	Plant	Barbed skullcup herb	Scutellaria barbata D.Don	Whole plant	2.372
41	Plant	Barberry	Berberis vulgaris L.	Fruit	0.011
42	Plant	Bay leaf, Laurel	Laurus nobilis L.	Leaf	1.636
43	Plant	Bearberry leaf	Arctostaphylos uva-ursi (L.) Spreng	Leaf	0.735
44	Plant	Beautiful sweetgum fruit	Liquidambar formosana Hance	Fruit	0.778
45	Plant	Beet	Beta vulgaris L.	Root	0.124
46	Plant	Belvedere fruit	Bassia scoparia (L.) A.J.Scott	Fruit	7.322
47	Plant	Bilberry	Vaccinium myrtillus L.	Leaf	0.397
48	Plant	Birch, Abedul, Betula	Betula pendula Roth	Leaf	0.132
49	Plant	Bitter bottle gourd	Cucurbita pepo L.	Fruit	0.151
50	Plant	Bitter melon	Momordica charantia L.	Fruit	118.514
51	Plant	Bitter orange peel	Citrus × aurantium L.	Fruit (Peel)	0.050
52	Plant	Black tea	Camellia sinensis (L.) Kuntze	Leaf	1.075
53	Plant	Blackthorn	Prunus spinosa L.	Fruit	0.175
54	Plant	Bladder wrack	Fucus evanescens C.Agardh	Whole plant	4.945
55	Plant	Boldo, Boldus	Peumus boldus Molina	Leaf	0.156
56	Plant	Boston ivy, Japanese ivy	Parthenocissus tricuspidata (Siebold & Zucc.) Planch	Leaf	96.453
57	Plant	Brown rice	Oryza sativa L.	Seed	3.446
58	Plant	Bupleurum root	Bupleurum falcatum L.	Root	148.514
59	Plant	Burdock	Arctium lappa L.	Root	4.295
60	Plant	Burdock fruit	Arctium lappa L.	Fruit	8.577
61	Plant	Cabbage	Brassica oleracea L.	Leaf	0.257
62	Plant	Calendula, Marigold	Calendula officinalis L.	Flower	21.622
63	Plant	Calumba	Jateorhiza palmata (Lam.) Miers	Root	4.549
64	Plant	Caraway	Carum carvi L.	Fruit	2.004
65	Plant	Cardamon	Elettaria cardamomum (L.) Maton	Fruit	9.203
66	Plant	Carob, St. john’s bread	Ceratonia siliqua L.	Fruit (Bean pod)	0.009
67	Plant	Cassia seed	Senna obtusifolia (L.) H.S.Irwin & Barneby	Seed	0.020
68	Plant	Cassis, Black currant	Ribes nigrum L.	Fruit	0.033
69	Plant	Cassis, Black currant	Ribes nigrum L.	Leaf	26.649
70	Plant	Catalpa fruit	Catalpa ovata G.Don	Fruit	2.199
71	Plant	Catnip, Catmint	Nepeta cataria L.	Stem and Leaf	13.009
72	Plant	Cat’s whisker, Java tea	Orthosiphon aristatus (Blume) Miq	Leaf	11.472
73	Plant	Cauliflower	Brassica oleracea L.	Stem	0.128
74	Plant	Celandine	Chelidonium majus L.	Whole plant	32.824
75	Plant	Celery	Apium graveolens L.	Root	0.037
76	Plant	Celery seed	Apium graveolens L.	Seed	4.377
77	Plant	Chaenomeles fruit	Pseudocydonia sinensis (Dum.Cours.) C.K.Schneid	Fruit	3.088
78	Plant	Chamaecrista herb	Chamaecrista nomame (Makino) H.Ohashi	Whole plant	0.833
79	Plant	Chaste tree	Vitex agnus-castus L.	Fruit	4.124
80	Plant	Cherry bark	Prunus jamasakura (Makino) Siebold ex Koidz	Stem (Bark)	1.300
81	Plant	China berry	Melia azedarach L./Melia azedarach var. subtripinnata Miq	Leaf	0.331
82	Plant	Chinese blackberry, sweet tea	Rubus chingii var. suavissimus (S.K.Lee) L.T.Lu	Leaf	0.304
83	Plant	Chinese honeylocust spine	Gleditsia sinensis Lam.	Stem (Hook)	84.297
84	Plant	Chinese prickly ash	Zanthoxylum simulans Hance	Fruit (Peel)	13.399
85	Plant	Chinese pulsatilla root	Pulsatilla chinensis (Bunge) Regel	Root	58.676
86	Plant	Chokeberry	Aronia melanocarpa (Michx.) Elliott	Fruit	0.106
87	Plant	Chrysanthemum flower	Chrysanthemum indicum L.	Flower (Capitula)	13.399
88	Plant	Chundan, Kathala hibutu tea	Salacia reticulata Wight	Root (Root bark)	0.678
89	Plant	Cimicifuga rhizome	Actaea dahurica (Turcz. ex Fisch. & C.A.Mey.) Franch	Root (Rhizome)	13.349
90	Plant	Cimicifuga rhizome	Actaea simplex (DC.) Wormsk. ex Prantl	Root (Rhizome)	4.822
91	Plant	Cinnamon	Neolitsea cassia (L.) Kosterm	Stem (Bark)	0.047
92	Plant	Cinnamon bark (Crude drug)	Neolitsea cassia (L.) Kosterm	Stem (Bark)	12.386
93	Plant	Citrus peel	Citrus × aurantium L./Citrus reticulata Blanco	Fruit (Peel)	0.106
94	Plant	Citrus unshiu peel	Citrus × aurantium f. deliciosa (Ten.) M.Hiroe/Citrus reticulata Blanco	Fruit (Peel)	0.133
95	Plant	Clematis root	Clematis terniflora var. mandshurica (Rupr.) Ohwi	Root	2.953
96	Plant	Clove (Crude drug)	Syzygium aromaticum (L.) Merr. & L.M.Perry	Flower (Flower bud)	0.216
97	Plant	Clove	Syzygium aromaticum (L.) Merr. & L.M.Perry	Flower (Flower bud)	0.075
98	Plant	Club moss	Lycopodium clavatum L.	Whole plant	2.434
99	Plant	Cnidium monnieri fruit	Cnidium monnieri (L.) Cusson	Fruit	36.730
100	Plant	Cnidium rhizome	Ligusticum officinale (Makino) Kitag	Root (Rhizome)	5.264
101	Plant	Codonopsis root	Codonopsis pilosula (Franch.) Nannf	Root	0.322
102	Plant	Cola	Cola nitida (Vent.) Schott & Endl	Seed	0.121
103	Plant	Coltsfoot	Tussilago farfara L.	Leaf	2.162
104	Plant	Coltsfoot flower	Tussilago farfara L.	Flower (Flower bud)	1.916
105	Plant	Comfrey, Boneset	Symphytum officinale L.	Root	29.459
106	Plant	Comfrey, Boneset	Symphytum officinale L.	Whole plant	21.986
107	Plant	Common Curculigo rhizome	Curculigo orchioides Gaertn	Root (Rhizome)	0.778
108	Plant	Common ducksmeat herb	Spirodela polyrhiza (L.) Schleid	Whole plant	366.554
109	Plant	Common knotgrass herb	Polygonum aviculare L.	Whole plant	14.243
110	Plant	Common mullein, Great mullein	Verbascum thapsus L.	Stem and Leaf	2.649
111	Plant	Common reed	Phragmites australis (Cav.) Trin. ex Steud	Stem	6.554
112	Plant	Coptis rhizome	Coptis japonica (Thunb.) Makino	Root (Rhizome)	0.289
113	Plant	Coriander	Coriandrum sativum L.	Fruit	20.986
114	Plant	Corn silk	Zea mays L.	Flower (Flower’s style)	180.068
115	Plant	Cornflower	Centaurea cyanus L.	Flower	0.155
116	Plant	Cornus fruit	Cornus officinalis Siebold & Zucc	Fruit	0.043
117	Plant	Corydalis tuber	Corydalis yanhusuo (Y.H.Chou & Chun C.Hsu) W.T.Wang ex Z.Y.Su & C.Y.Wu	Root (Tuber)	8.796
118	Plant	Couch grass, Quack grass	Elymus repens (L.) Gould	Root (Rhizome)	392.635
119	Plant	Cowherb	Gypsophila vaccaria (L.) Sm	Seed	0.354
120	Plant	Crataegus fruit	Crataegus cuneata Siebold & Zucc	Fruit	0.244
121	Plant	Cumin	Cuminum cyminum L.	Fruit	80.054
122	Plant	Curcuma rhizome	Curcuma zedoaria (Christm.) Roscoe	Root (Rhizome)	65.608
123	Plant	Cyperus rhizome	Cyperus rotundus L.	Root (Rhizome)	2.801
124	Plant	Daisy fleabane	Erigeron annuus (L.) Pers	Whole plant	0.389
125	Plant	Damiana	Turnera diffusa Willd. ex Schult	Leaf	1.962
126	Plant	Dandelion	Taraxacum Weber	Root	4.945
127	Plant	Dayflower	Commelina communis L.	Whole plant	6.058
128	Plant	Devil’s claw	Harpagophytum procumbens (Burch.) DC. ex Meisn	Root (Tuber)	0.188
129	Plant	Dioscorea rhizome	Dioscorea japonica Thunb.	Root (Rhizome)	0.010
130	Plant	Dipsacus root	Dipsacus asper Wall. ex DC	Root	0.170
131	Plant	Echinacea	Echinacea purpurea (L.) Moench	Stem and Leaf	10.635
132	Plant	Elder	Sambucus nigra L.	Flower	9.359
133	Plant	English hawthorn	Crataegus laevigata (Poir.) DC	Leaf	36.730
134	Plant	Ephedra herb	Ephedra sinica Stapf	Stem	0.723
135	Plant	Epimedium herb	Epimedium grandiflorum var. koreanum (Nakai) K.Suzuki	Leaf	11.359
136	Plant	Erythrina bark	Erythrina variegata L.	Stem (Bark)	16.959
137	Plant	Eucalyptus	Eucalyptus globulus Labill.	Leaf	0.023
138	Plant	Eucommia bark	Eucommia ulmoides Oliv.	Stem (Bark)	0.188
139	Plant	Eucommia leaf	Eucommia ulmoides Oliv.	Leaf	2.791
140	Plant	Euodia fruit	Tetradium ruticarpum (A.Juss.) T.G.Hartley	Fruit	10.642
141	Plant	European verbena herb	Verbena officinalis L.	Stem	7.001
142	Plant	Eyebright	Euphrasia officinalis L.	Stem and Leaf	8.577
143	Plant	Feather cockscomb seed	Celosia argentea L.	Seed	2.791
144	Plant	Fennel	Foeniculum vulgare Mill.	Fruit	0.045
145	Plant	Fenugreek	Trigonella foenum-graecum L.	Stem and Leaf	36.351
146	Plant	Fermented black soybean	Glycine max (L.) Merr.	Seed	5.264
147	Plant	Feverfew	Tanacetum parthenium (L.) Sch.Bip.	Whole plant	8.714
148	Plant	Figwort flower Picrorhiza rhizome	Neopicrorhiza scrophulariiflora (Pennell) D.Y.Hong	Root (Rhizome)	1.678
149	Plant	Finger citron	Citrus medica L.	Fruit (Peel)	18.716
150	Plant	Flatstem milkvetch seed	Phyllolobium chinense Fisch.	Seed	4.295
151	Plant	Forsythia fruit	Forsythia suspensa (Thunb.) Vahl.	Fruit	10.530
152	Plant	Fortune windmill palm petiole	Trachycarpus fortunei (Hook.) H.Wendl.	Leaf	1.151
153	Plant	Fortune’s drynaria rhizome	Drynaria roosii Nakaike	Root (Rhizome)	423.041
154	Plant	Foxtail millet	Setaria italica (L.) P.Beauv.	Seed	0.006
155	Plant	Fragrant rosewood	Dalbergia odorifera T.C.Chen	Root (Heart wood)	1.418
156	Plant	Fragrant solomonseal rhizome	Polygonatum odoratum (Mill.) Druce	Root (Rhizome)	0.894
157	Plant	Frankincense	Boswellia sacra Flück.	Resin	0.003
158	Plant	French bean	Phaseolus vulgaris L.	Fruit (Bean pod)	0.936
159	Plant	Garden burnet root	Sanguisorba officinalis L.	Root (Root and Rhizome)	6.392
160	Plant	Gardenia fruit	Gardenia jasminoides J.Ellis	Fruit	0.014
161	Plant	Gastrodia tuber	Gastrodia elata Blume	Root (Tuber)	0.155
162	Plant	Gentiana macrophylla root	Gentiana macrophylla Pall.	Root	0.385
163	Plant	Geranium herb	Geranium thunbergii Siebold & Zucc. ex Lindl. & Paxton	Stem and Leaf	10.642
164	Plant	German chamomile	Matricaria chamomilla L.	Flower	0.322
165	Plant	Ginger	Zingiber officinale Roscoe	Root (Rhizome)	122.020
166	Plant	Ginkgo	Ginkgo biloba L	Leaf	6.936
167	Plant	Ginseng	Panax ginseng C.A.Mey	Root	0.023
168	Plant	Glechoma hederacea herb	Glechoma grandis (A.Gray) Kuprian.	Stem and Leaf	3.470
169	Plant	Glycyrrhiza	Glycyrrhiza uralensis Fisch. ex DC.	Root	3.101
170	Plant	Goldenrod	Solidago virgaurea subsp. asiatica (Nakai ex Hara) Kitam. ex Hara L.	Leaf	18.378
171	Plant	Gooseberry, European gooseberry	Ribes uva-crispa L.	Fruit	0.000
172	Plant	Gorgon euryale seed	Euryale ferox Salisb.	Seed	0.081
173	Plant	Grape	Vitis L.	Leaf	1.151
174	Plant	Green tea leaf	Camellia sinensis (L.) Kuntze.	Leaf	0.115
175	Plant	Guarana seed	Paullinia cupana Kunth.	Seed	5.058
176	Plant	Guava	Psidium guajava L.	Fruit	0.080
177	Plant	Gymnema	Gymnema sylvestre (Retz.) R.Br. ex Sm.	Leaf	0.346
178	Plant	Haichow Elsholtzia herb	Elsholtzia splendens var. splendens	Whole plant	3.839
179	Plant	Hairyveine agrimonia herb	Agrimonia pilosa Ledeb.	Whole plant	23.892
180	Plant	Heather	Calluna vulgaris (L.) Hull.	Flower (Flower bud)	4.124
181	Plant	Hedysarum root	Hedysarum polybotrys Hand.-Mazz.	Root	1.364
182	Plant	Henna	Lawsonia inermis L.	Leaf	1.104
183	Plant	Heterophylly false starwort root	Pseudostellaria heterophylla (Miq.) Pax.	Root (Tuberous root)	0.141
184	Plant	Hibiscus, Roselle	Hibiscus sabdariffa L.	Flower (Calyx)	0.030
185	Plant	Hollyhock	Alcea rosea L.	Flower	0.906
186	Plant	Hop strobile	Humulus lupulus L.	Flower	1.628
187	Plant	Horse chestnut	Aesculus hippocastanum L.	Leaf	0.397
188	Plant	Horseradish	Armoracia rusticana G.Gaertn., B.Mey. & Scherb.	Root	0.422
189	Plant	Horsetail, Field hare-tail	Equisetum arvense L.	Stem	0.639
190	Plant	Houttuynia herb	Houttuynia cordata Thunb.	Whole plant (Aerial part)	1.880
191	Plant	Hovenia seed or fruit	Hovenia dulcis Thunb.	Fruit	3.446
192	Plant	Hyssop	Hyssopus officinalis L.	Stem and Leaf	4.945
193	Plant	Immature citrus unshiu peel	Citrus × aurantium f. deliciosa (Ten.) M.Hiroe/Citrus reticulata Blanco	Fruit (Peel)	0.091
194	Plant	Immature orange fruit	Citrus × aurantium L.	Fruit	0.066
195	Plant	Indian madder root	Rubia cordifolia L.	Root	12.147
196	Plant	Indigo	Isatis tinctoria L.	Branch and Leaf	9.762
197	Plant	Inula flower	Pentanema britannica (L.) D.Gut.Larr., Santos-Vicente, Anderb., E.Rico & M.M.Mart.Ort	Flower (Capitula)	13.349
198	Plant	Ipe, Taheebo	Handroanthus impetiginosus (Mart. ex DC.) Mattos	Stem (Bark)	4.377
199	Plant	Ipecac	Carapichea ipecacuanha (Brot.) L.Andersson	Root	0.577
200	Plant	Isatis root	Isatis tinctoria L.	Root	0.373
201	Plant	Isodon herb	Isodon japonicus (Burm.f.) H.Hara	Stem and Leaf	5.270
202	Plant	Japanese angelica root	Angelica acutiloba (Siebold & Zucc.) Kitag.	Root	16.284
203	Plant	Japanese angelica tree	Aralia elata (Miq.) Seem	Stem	2.791
204	Plant	Japanese banana, Hardy banana	Musa basjoo Siebold & Zucc. ex Iinuma	Root	94.135
205	Plant	Japanese bush cherry	Prunus japonica Thunb.	Seed	1.300
206	Plant	Japanese gentian	Gentiana scabra Bunge.	Root (Root and Rhizome)	2.791
207	Plant	Japanese horse chestnut	Aesculus turbinata Blume.	Fruit	0.098
208	Plant	Japanese primrose	Primula sieboldii É.Morren	Flower	51.176
209	Plant	Japanese thistle root	Cirsium japonicum DC	Root	13.009
210	Plant	Japanese thyme	Thymus quinquecostatus Čelak	Leaf	11.359
211	Plant	Japanese torreya	Torreya nucifera (L.) Siebold & Zucc.	Seed	0.094
212	Plant	Japanese valerian	Valeriana fauriei Briq.	Root (Root and Rhizome)	9.203
213	Plant	Jasmine	Jasminum L.	Flower	0.044
214	Plant	Javanese turmeric	Curcuma zanthorrhiza Roxb.	Root (Rhizome)	13.399
215	Plant	Jujube	Ziziphus jujuba var. inermis (Bunge) Rehder	Fruit	0.123
216	Plant	Jujube seed	Ziziphus jujuba var. spinosa (Bunge) Hu ex H.F.Chow	Seed	0.002
217	Plant	Juniper berry	Juniperus communis L.	Fruit	0.019
218	Plant	Kava, Kava pepper	Piper methysticum G.Forst	Root	54.365
219	Plant	Kidachi aloe	Aloe arborescens Mill.	Leaf	1.174
220	Plant	Kombu	Laminariaceae	Root	1.117
221	Plant	Kuwagataso	Veronica miqueliana Nakai	Stem and Leaf	7.481
222	Plant	Lady’s mantle	Alchemilla vulgaris L.	Leaf	6.936
223	Plant	Lemon grass	Cymbopogon citratus (DC.) Stapf.	Stem and Leaf	0.142
224	Plant	Licorice	Glycyrrhiza glabra L.	Root	22.730
225	Plant	Ligusticum sinense rhizome	Conioselinum anthriscoides (H.Boissieu) Pimenov & Kljuykov	Root (Rhizome)	10.642
226	Plant	Ligustrum fruit	Ligustrum lucidum W.T.Aiton	Fruit	8.430
227	Plant	Lilium bulb	Lilium lancifolium Thunb.	Leaf (Bulb)	0.056
228	Plant	Linden, Lime flower	Tilia cordata Mill.	Leaf	3.891
229	Plant	Lindera root	Lindera aggregata var. aggregata	Root	61.986
230	Plant	Lithospermum root	Lithospermum erythrorhizon Siebold & Zucc.	Root	10.530
231	Plant	Long pepper	Piper longum L.	Fruit	1.063
232	Plant	Lonicera flower	Lonicera japonica Thunb.	Flower (Flower bud)	0.256
233	Plant	Lonicera leaf and stem	Lonicera japonica Thunb.	Stem and Leaf	2.162
234	Plant	Lophatherum herb	Lophatherum gracile Brongn.	Whole plant	1.628
235	Plant	Loquat leaf	Eriobotrya japonica (Thunb.) Lindl.	Leaf	0.084
236	Plant	Low-bush cranberry	Vaccinium macrocarpon Aiton	Leaf	3.268
237	Plant	Luffa, Vegetable sponge	Luffa aegyptiaca Mill.	Fruit (Fiber of mature fruit)	23.892
238	Plant	Luobuma	Apocynum venetum L.	Leaf	5.984
239	Plant	Lycium bark	Lycium chinense Mill.	Root (Root bark)	5.609
240	Plant	Lycium leaf	Lycium chinense Mill.	Branch and Leaf	6.155
241	Plant	Magnolia bark	Magnolia obovata Thunb.	Stem (Bark)	2.946
242	Plant	Magnolia flower	Magnolia kobus DC	Flower (Flower bud)	0.131
243	Plant	Mallotus bark	Mallotus japonicus (L.f.) Müll.Arg.	Stem (Bark)	3.088
244	Plant	Mallow	Malva sylvestris L.	Whole plant	5.469
245	Plant	Malt	Hordeum vulgare L.	Seed	73.500
246	Plant	Marjoram, Sweet marjoram	Origanum majorana L.	Leaf	0.141
247	Plant	Marshmallow, Altea	Althaea officinalis L.	Leaf	19.486
248	Plant	Marshmallow, Altea	Althaea officinalis L.	Root	8.276
249	Plant	Meadowsweet	Filipendula ulmaria (L.) Maxim	Stem and Leaf	26.486
250	Plant	Melia fruit	Melia azedarach L.	Fruit (Mature fruit)	0.049
251	Plant	Melilot	Melilotus officinalis (L.) Lam.	Whole plant	2.503
252	Plant	Mentha herb	Mentha canadensis L.	Leaf	6.199
253	Plant	Milk thistle	Silybum marianum (L.) Gaertn.	Whole plant	2.059
254	Plant	Mountain ash	Sorbus aucuparia L.	Fruit	0.121
255	Plant	Moutan bark	Paeonia × suffruticosa Andrews	Root (Root bark)	1.424
256	Plant	Mulberry	Morus alba L.	Leaf	3.101
257	Plant	Mulberry bark	Morus alba L.	Root (Root bark)	10.530
258	Plant	Mulberry fruit	Morus alba L.	Fruit	5.264
259	Plant	Myrobalan fruit	Terminalia chebula Retz.	Fruit	0.001
260	Plant	Nandina fruit	Nandina domestica Thunb.	Fruit	2.649
261	Plant	Natural indigo (Dye)	Indigofera tinctoria L.	Leaf	0.020
262	Plant	Nikko maple	Acer maximowiczianum Miq.	Stem (Bark)	0.906
263	Plant	Notopterygium	Hansenia weberbaueriana (Fedde ex H.Wolff) Pimenov & Kljuykov	Root (Rhizome)	2.946
264	Plant	Nutmeg	Myristica fragrans Houtt.	Seed	0.012
265	Plant	Oat	Avena sativa L.	Stem and Leaf	825.541
266	Plant	Olive	Olea europaea L.	Leaf	0.302
267	Plant	Ophiopogon root	Ophiopogon japonicus (Thunb.) Ker Gawl.	Root	0.220
268	Plant	Orange daylily	Hemerocallis fulva var. fulva	Flower (Flower bud)	0.022
269	Plant	Orange flower	Citrus × aurantium L.	Flower	0.919
270	Plant	Orange leaf	Citrus × aurantium L.	Leaf	30.878
271	Plant	Orange peel (bitter)	Citrus × aurantium L.	Fruit	0.015
272	Plant	Oregano	Origanum vulgare L.	Stem and Leaf	7.350
273	Plant	Oriental arborvitae leafy twig	Platycladus orientalis (L.) Franco.	Leaf	3.470
274	Plant	Orris root	Iris florentina L.	Root	12.327
275	Plant	Pale butterfly bush flower	Buddleja officinalis Maxim.	Flower (Flower bud)	0.208
276	Plant	Panax japonicus rhizome	Panax japonicus (T.Nees) C.A.Mey	Root (Rhizome)	0.322
277	Plant	Panax notoginseng root	Panax notoginseng (Burkill) F.H.Chen	Root	0.002
278	Plant	Parsley	Petroselinum crispum subsp. crispum	Leaf	0.627
279	Plant	Patrinia herb	Patrinia scabiosifolia Link	Whole plant	0.723
280	Plant	Peach	Prunus persica (L.) Batsch	Leaf	0.464
281	Plant	Pennyroyal	Mentha pulegium L.	Whole plant	0.389
282	Plant	Peony	Paeonia lactiflora Pall.	Flower	0.125
283	Plant	Peony root	Paeonia lactiflora Pall.	Root	0.529
284	Plant	Perilla fruit	Perilla frutescens var. frutescens	Fruit	0.084
285	Plant	Perilla herb	Perilla frutescens var. crispa (Thunb.) H.Deane	Leaf	4.650
286	Plant	Perilla, Beefsteak plant	Perilla frutescens (L.) Britton	Stem	5.264
287	Plant	Persimmon	Diospyros kaki L.f.	Leaf	1.474
288	Plant	Persimmon calyx	Diospyros kaki L.f.	Fruit (Calyx)	0.465
289	Plant	Peucedanum root	Kitagawia praeruptora (Dunn) Pimenov.	Root	1.628
290	Plant	Phellodendron bark	Phellodendron amurense Rupr.	Stem (Bark)	6.993
291	Plant	Pine	Pinus L.	Leaf	1.019
292	Plant	Pinellia tuber	Pinellia ternata (Thunb.) Makino	Root (Tuber)	1.872
293	Plant	Plantago herb	Plantago asiatica L.	Whole plant	2.642
294	Plant	Plantago seed	Plantago asiatica L.	Seed	0.385
295	Plant	Platycodon root	Platycodon grandiflorus (Jacq.) A.DC.	Root	2.199
296	Plant	Polygala root	Polygala tenuifolia Willd.	Root	0.075
297	Plant	Polygonatum rhizome	Polygonatum falcatum A.Gray	Root (Rhizome)	0.006
298	Plant	Polygonum root	Reynoutria multiflora (Thunb.) Moldenke	Root (Tuberous root)	0.008
299	Plant	Pomegranate rind	Punica granatum L.	Fruit (Peel)	0.777
300	Plant	Potentilla, Silverweed	Argentina anserina (L.) Rydb	Whole plant	6.731
301	Plant	Prickly pear cactus	Opuntia Mill.	Flower	1.174
302	Plant	Processed ginger	Zingiber officinale Roscoe	Root (Rhizome)	45.486
303	Plant	Processed mume	Prunus mume (Siebold) Siebold & Zucc.	Fruit	0.010
304	Plant	Prunella spike	Prunella vulgaris subsp. asiatica (Nakai) H.Hara	Flower (Spike)	35.216
305	Plant	Psoralea corylifolia fruit	Cullen corylifolium (L.) Medik.	Fruit	10.605
306	Plant	Pueraria Root	Pueraria montana var. lobata (Willd.) Maesen & S.M.Almeida ex Sanjappa & Predeep	Root	7.547
307	Plant	Purslane herb	Portulaca oleracea L.	Whole plant	13.055
308	Plant	Quercus bark	Quercus acutissima Carruth.	Stem (Bark)	6.836
309	Plant	Quercus salicina leaf	Quercus salicina Blume.	Leaf	0.259
310	Plant	Raspberry	Rubus idaeus L.	Leaf	9.614
311	Plant	Red clover	Trifolium pratense L.	Whole plant	7.507
312	Plant	Red poppy, Corn poppy	Papaver rhoeas L.	Flower	5.001
313	Plant	Rehmannia root	Rehmannia glutinosa (Gaertn.) DC	Root	0.529
314	Plant	Rhubarb	Rheum palmatum L.	Root (Rhizome)	1.364
315	Plant	Rice paper plant	Tetrapanax papyrifer (Hook.) K.Koch	Stem (Pith)	0.028
316	Plant	Rooibos	Aspalathus linearis (Burm.f.) R.Dahlgren	Leaf	94.135
317	Plant	Rose	Rosa L.	Flower (Flower bud)	0.028
318	Plant	Rose fruit	Rosa multiflora Thunb.	Fruit	2.196
319	Plant	Rosehips	Rosa L.	Fruit (Peel)	0.030
320	Plant	Rosemary	Salvia rosmarinus Spenn.	Leaf	1.555
321	Plant	Rosewood	Dalbergia cochinchinensis Pierre	Stem (Heart wood)	0.004
322	Plant	Rugosa rose flower	Rosa rugosa Thunb.	Flower (Flower bud)	0.156
323	Plant	Sacred lotus, Lotus	Nelumbo nucifera Gaertn.	Root (Rhizome node)	624.459
324	Plant	Safflower	Carthamus tinctorius L.	Flower	38.392
325	Plant	Salvia miltiorrhiza root	Salvia miltiorrhiza Bunge.	Root	10.530
326	Plant	Sambucus wood	Sambucus williamsii Hance.	Stem	2.515
327	Plant	Saposhnikovia root and rhizome	Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk	Root (Root and Rhizome)	4.650
328	Plant	Sappan wood	Biancaea sappan (L.) Tod.	Stem (Heart wood)	0.198
329	Plant	Sargentgloryvine stem	Sargentodoxa cuneata (Oliv.) Rehder & E.H.Wilson	Stem	1.488
330	Plant	Sarsaparilla	Smilax purhampuy Ruiz.	Root (Root and Rhizome)	24.176
331	Plant	Saussurea root	Dolomiaea costus (Falc.) Kasana & A.K.Pandey	Root	5.609
332	Plant	Schizonepeta spike	Nepeta tenuifolia Benth.	Flower (Spike)	0.596
333	Plant	Scisandra fruit	Schisandra chinensis (Turcz.) Baill	Fruit	0.022
334	Plant	Scrophularia root	Scrophularia ningpoensis Hemsl.	Root	1.916
335	Plant	Scutellaria root	Scutellaria baicalensis Georgi	Root	2.485
336	Plant	Sea buckthorn, Argasse	Hippophae rhamnoides L.	Fruit	0.024
337	Plant	Seaweed	Sargassum fusiforme (Harvey) Setchell	Whole plant	2.668
338	Plant	Senna Leaf	Senna alexandrina var. alexandrina	Leaf	0.206
339	Plant	Sesame	Sesamum indicum L.	Seed	2.004
340	Plant	Sheep sorrel	Rumex acetosella subsp. pyrenaicus (Pourr. ex Lapeyr.) Akeroyd	Whole plant	6.392
341	Plant	Shiny bugleweed	Lycopus lucidus Turcz. ex Benth.	Stem and Leaf	1.364
342	Plant	Siberian cocklebur fruit	Xanthium strumarium L.	Fruit	0.098
343	Plant	Siberian ginseng	Eleutherococcus senticosus (Rupr. & Maxim.) Maxim.	Root	35.216
344	Plant	Silktree Albizia bark	Albizia julibrissin Durazz.	Stem (Bark)	0.927
345	Plant	Sinomenium stem	Sinomenium acutum (Thunb.) Rehder et E.H.Wilson	Stem	9.065
346	Plant	Smilax rhizome	Smilax glabra Roxb.	Root (Rhizome)	0.047
347	Plant	Snowbell-leaf tickclover herb	Grona styracifolia (Osbeck) H.Ohashi & K.Ohashi	Stem and Leaf	13.399
348	Plant	Songaria cynomorium herb	Cynomorium coccineum subsp. songaricum (Rupr.) J.Léonard	Stem (Fleshy stem)	0.075
349	Plant	Sophora japonica flower	Styphnolobium japonicum (L.) Schott.	Flower (Flower bud)	7.350
350	Plant	Sophora root	Sophora flavescens Aiton	Root	4.822
351	Plant	Sophora subprostrata root	Sophora tonkinensis var. tonkinensis	Root	7.001
352	Plant	Sour cherry	Prunus cerasus L.	Fruit	1.608
353	Plant	Sparganium rhizome	Sparganium stoloniferum (Buch.-Ham. ex Graebn.) Buch.-Ham. ex Juz.	Root (Rhizome)	0.018
354	Plant	Spatholobus suberectus stem	Spatholobus suberectus Dunn.	Stem (Vine)	0.927
355	Plant	Spearmint	Mentha spicata L.	Whole plant (Aerial part)	2.389
356	Plant	Spicebush	Lindera umbellata Thunb.	Stem	0.168
357	Plant	Spreading Hedyotis herb	Scleromitrion diffusum (Willd.) R.J.Wang	Whole plant	36.730
358	Plant	Star anise	Illicium verum Hook.f.	Fruit	0.010
359	Plant	Stellaria herb	Stellaria media (L.) Vill. L.	Whole plant	22.730
360	Plant	Stevia	Stevia rebaudiana (Bertoni) Bertoni	Whole plant	1.488
361	Plant	Stinging nettle, Nettle	Urtica dioica L.	Leaf	61.986
362	Plant	Summer savory	Satureja hortensis L.	Leaf	2.649
363	Plant	Sunflower	Helianthus annuus L.	Flower	0.120
364	Plant	Sweet flag root	Acorus calamus L.	Root (Rhizome)	1.364
365	Plant	Sweet hydrangea leaf	Hydrangea serrata (Thunb.) Ser.	Leaf	0.853
366	Plant	Sweet tea vine, Gospel herb	Gynostemma pentaphyllum (Thunb.) Makino	Stem	0.399
367	Plant	Sweet violet	Viola odorata L.	Whole plant	61.986
368	Plant	Sweet woodruff	Galium odoratum (L.) Scop.	Leaf	9.203
369	Plant	Sweet wormwood herb	Artemisia annua L.	Whole plant (Aerial part)	1.138
370	Plant	Tansy	Tanacetum vulgare L.	Whole plant	14.878
371	Plant	Tarragon	Artemisia dracunculus L.	Leaf	2.860
372	Plant	Tetragonia herb	Tetragonia tetragonoides (Pall.) Kuntze.	Whole plant	18.716
373	Plant	Tokoro rhizome	Dioscorea tokoro Makino ex Miyabe	Root (Rhizome)	3.107
374	Plant	Tokyo violet herb	Viola philippica var. philippica	Whole plant	11.377
375	Plant	Tribulus fruit	Tribulus terrestris L.	Fruit	1.104
376	Plant	Trichosanthes fruit	Trichosanthes kirilowii Maxim.	Fruit	0.238
377	Plant	Trichosanthes peel	Trichosanthes kirilowii Maxim.	Fruit (Peel)	2.418
378	Plant	Trichosanthes root	Trichosanthes kirilowii Maxim.	Root	0.180
379	Plant	Trichosanthes seed	Trichosanthes kirilowii Maxim.	Seed	0.029
380	Plant	Trifoliate orange, Hardy orange	Citrus trifoliata L.	Fruit	0.058
381	Plant	Turmeric	Curcuma longa L.	Root (Rhizome)	21.095
382	Plant	Uncaria hook	Uncaria rhynchophylla (Miq.) Miq.	Stem (Hook)	3.891
383	Plant	Violet	Viola L.	Stem and Leaf	2.059
384	Plant	Walnut	Juglans L.	Fruit (Hull)	11.472
385	Plant	Walnut	Juglans L.	Leaf	6.561
386	Plant	Walnut	Juglans regia L.	Seed	0.033
387	Plant	Water chestnut	Trapa natans var. bispinosa (Roxb.) Makino	Fruit	2.953
388	Plant	Watercress	Nasturtium officinale R.Br.	Stem	20.041
389	Plant	Wheat	Triticum aestivum L.	Seed	0.244
390	Plant	White dead-nettle	Lamium album subsp. barbatum (Siebold & Zucc.) Mennema	Stem and Leaf	25.365
391	Plant	White horehound	Marrubium vulgare L.	Whole plant	7.782
392	Plant	White sandalwood	Santalum album L.	Stem (Xylem)	0.176
393	Plant	White willow	Salix alba L.	Stem (Bark)	0.373
394	Plant	Wild strawberry	Fragaria vesca L.	Leaf	2.286
395	Plant	Witch hazel, Hamamelis	Hamamelis virginiana L.	Leaf	1.256
396	Plant	Wormwood, Mugwort	Artemisia princeps Pamp.	Whole plant	2.059
397	Plant	Yarrow	Achillea millefolium L.	Whole plant	1.306
398	Plant	Yerbadetajo herb	Eclipta prostrata (L.) L.	Stem and Leaf	6.155
399	Plant	Yew	Taxus brevifolia Nutt.	Leaf	0.487
400	Fungus	Agaricus	Agaricus blazei Murill.	Fruit body	0.005
401	Fungus	Baikisei, Artist’s bracket	Ganoderma applanatum (Pers.) Pat.	Fruit body	12.312
402	Fungus	Ganoderma	Ganoderma lucidum P.Karsten	Fruit body	0.004
403	Fungus	Iceland moss	Cetraria islandica (L.) Ach.	Lichen thallus	15.527
404	Fungus	Jelly ear	Auricularia auricula-judae (Bull.) Quél	Fruit body	38.392
405	Fungus	Meshima	Tropicoporus linteus (Berk. & M.A.Curtis) L.W.Zhou & Y.C.Dai	Fruit body	4.945
406	Fungus	Polyporus sclerotium	Polyporus umbellatus Fries	Sclerotium	3.673
407	Fungus	Poria sclerotium	Wolfiporia cocos Ryvarden & Gilbertson (Poria cocos Wolf)	Sclerotium	0.002
408	Fungus	Snow tea	Thamnolia vermicularis (Sw.) Ach. ex Schaer	Lichen thallus	0.422
409	Fungus	Turkey tail	Trametes versicolor (L.) Lloyd.	Fruit body	11.472
410	Other (non-plant)	Abalone shell	Haliotis diversicolor Reeve, 1846	Shell	0.778
411	Other (non-plant)	Earthworm	Pheretima aspergillum Perrier	Whole body	18.716
412	Other (non-plant)	Spirulina	Arthrospira platensis Gomont.	Algae	39.662
413	Other (non-plant)	Trogopterus feces	Trogopterus xanthipes (Milne-Edwards)	Feces	28.014
414	Other (non-plant)	Water buffalo horn	Bubalus bubalis	Horn	0.000

Figure 1.

The distribution of the LPS concentration of the 414 samples measured using the Limulus reaction. The samples were divided into plants, fungi, and others. The plant samples were further categorized according to their parts. *p-value < 0.05 for Steel–Dwass test.

Table 2.

Twelve herbal extract samples with significantly higher LPS content than the other samples. The samples are listed in the order of high concentration.

Sample name	Scientific name	Part	Limulus activity (μg/g)
Oat	Avena sativa L.	Stem and leaf	825.5
Sacred lotus, Lotus	Nelumbo nucifera Gaertn.	Root	624.5
Aralia rhizome	Aralia cordata Thunb.	Root	504.0
Fortune’s drynaria rhizome	Drynaria roosii Nakaike	Root	423.0
Couch grass, Quack grass	Elytrigia repens (L.) Gould	Root	392.6
Common ducksmeat	Spirodela polyrhiza (L.) Schleid.	Leaf	366.6
Angelica dahurica root	Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav.	Root	266.6
Corn silk	Zea mays L.	Flower	180.1
Bupleurum root	Bupleurum falcatum L.	Root	148.5
Ginger	Zingiber officinale Roscoe.	Root	122
Artemisia leaf	Artemisia princeps Pamp.	Leaf	121.8
Bitter melon	Momordica charantia L.	Fruit	118.5

Measurement of the macrophage activation potential of the herbal extracts

Twelve herbal extract samples with LPS levels of ≥ 100 μg/g were tested for macrophage activation potential. Macrophage activation potential was assessed by measuring phagocytosis and nitric oxide (NO) production by stimulating RAW 264.7 cells with the herbal extracts. Stimulation using purified LPSp served as a positive control. Phagocytic activity was increased in all samples compared with that in the non-stimulated control group (Fig. 2). The phagocytosis ability of RAW 264.7 cells was increased when stimulated with Oat (Avena sativa L.), Sacred lotus (Nelumbo nucifera Gaertn.), Aralia rhizome (Aralia cordata Thunb.), Fortune’s drynaria rhizome (Drynaria roosii Nakaike), Couch grass (Elytrigia repens (L.) Gould), Angelica dahurica root (Angelica dahurica), Common ducksmeat (Spirodela polyrhiza (L.) Schleid.), Corn silk (Zea mays L.), and Bupleurum root (Bupleurum falcatum L.) compared with the positive control LPSp. The phagocytosis ability of RAW 264.7 cells with Ginger (Zingiber officinale Roscoe) was comparable, and that of Artemisia leaf (Artemisia princeps Pamp.) and Bitter melon (Momordica charantia L.) was lower than that of LPSp. The Pearson correlation between the amount of LPS and phagocytosis showed a clear positive correlation at R = 0.474. This suggests that LPS in crude drugs may increase the phagocytosis ability of macrophages, but other factors may also be involved.

Figure 2.

The percentage of phagocytic activity of RAW 264.7 cells stimulated by the 12 herbal extract samples containing the highest LPS levels are listed in Table 2. The concentrations of herbs and LPSp added were adjusted so that the LPS concentration was 100 ng/ml. The dotted line represents the phagocytosis percentage of RAW 264.7 cells without any external stimulation (medium only). Each bar represents the mean of two independent measurements, and the error bars represent the standard deviation.

To compare the NO production ability of the 12 herbal extracts with that of the positive control LPSp, the dose–response curves of the 12 herbal extract samples are presented in Fig. 3. The 12 herbal extracts were divided based on the amount of LPS required to induce 5 µM more nitrite than LPSp. Oat, (Avena sativa L.), Sacred lotus (Nelumbo nucifera Gaertn.), Fortune’s drynaria rhizome (Drynaria roosii Nakaike), and Couch grass (Elytrigia repens (L.) Gould) required a fewer samples per LPS content to induce 5 µM NO compared with LPSp (Fig. 3a). Corn silk (Zea mays L.), Bupleurum root (Bupleurum falcatum L.), Angelica dahurica root (Angelica dahurica), Common duckmeat (Spirodela polyrhiza (L.) Schleid.), and Angelica dahurica root (Angelica dahurica) required equivalent amounts of LPSp (Fig. 3b), whereas Ginger (Zingiber officinale Roscoe), Artemisia leaf (Artemisia princeps Pamp.), and Bitter melon (Momordica charantia L.) required more samples per LPS content to induce 5 µM Nitrite compared with LPSp (Fig. 3c). Table 3 shows the amount of LPS content in each herbal extract required to induce 5 µM NO and the relative NO induction strength compared with LPSp.

Figure 3.

Dose–response curve of macrophage activation capacity determined by measuring the amount of NO produced as the amount of nitrite produced by RAW 264.7 cells stimulated by adding 1, 10, and 100 ng/ml per LPS to the 12 herbal extract samples containing the highest LPS content listed in Table 2. The amount of LPS needed to induce 5 µM more nitrite than LPSp used as control is (a) less than LPSp, (b) equivalent to LPSp, and (c) more than LPSp in this group. The dotted lines represent 5 µM Nitrite. The trendline equations (dashed lines) and R² of each line are listed in Table 3.

Table 3.

The equivalent amount of herbal extracts per LPS content needed to induce 5 µM nitrite, which is the relative nitrite induction strength compared with LPSp. The trendline equations and R² of each line in Fig. 3 are also listed.

Samples	LPS (ng/mL)/nitrite 5 µM	Relative NO induction strength (LPSp)	Trendline equation	R2 value
LPSp	32.8	1.0	y = 1.6218x0.3226	1.00
Oat	7.3	4.5	y = 2.3505ln(x) + 0.3357	0.99
Sacred lotus	19.7	1.7	y = 1.0709x0.517	0.99
Aralia rhizome	24.7	1.3	y = 1.4364ln(x) + 0.3958	0.98
Fortune’s drynaria rhizome	14.3	2.3	y = 1.4994x0.4533	1.00
Couch grass	3.6	9.2	y = 2.7422ln(x) + 1.5183	1.00
Common Ducksmeat	14.6	2.2	y = 1.5147ln(x) + 0.937	0.99
Angelica dahurica root	18.3	1.8	y = 1.6192ln(x) + 0.2956	0.97
Corn silk	15.8	2.1	y = 1.6192ln(x) + 0.5361	1.00
Bupleurum root	12.0	2.7	y = 2.011ln(x) − 0.0051	0.98
Ginger	222.7	0.1	y = 0.903x0.3166	0.64
Artemisia leaf	145.2	0.2	y = 1.006x0.3221	0.73

NO production results suggested that herbal extracts containing high LPS levels can activate macrophages. Moreover, NO production was significantly inhibited by the reaction with polymyxin B, an LPS inhibitor. In addition, an LPS inhibitor was used by Montenegro et al. as a way to verify that NO-inducing activity is obtained from LPS. The 12 samples exhibited significant inhibition of NO production, with inhibition rates of 71–95% (Fig. 4). The decrease in NO production when polymyxin was added suggests that it is mostly the LPS content that is involved in the macrophage-activating capacity of these herbal extracts.

Figure 4.

Macrophage activation potential determined by measuring the NO production of RAW 264.7 cells stimulated by the 12 herbal samples containing the highest LPS levels listed in Table 2. The percentage of NO produced by RAW 264.7 cells stimulated by LPS content (black bars) and other components (white) in the herbal extract samples. The concentrations of herbs and LPSp added were adjusted, making the LPS content 10 ng/ml. The black area represents the percentage of induced NO₂ being decreased following polymyxin B addition, representing the percentage of NO₂ induced by the LPS content in the herb samples. Each bar represents the mean of two independent measurements, and the error bars represent the standard deviation.

Discussion

Herbal extracts have several health-benefiting effects, such as hemostatic^44,45, antifebrile^46,47, detoxifying⁴⁸, sweating⁴⁹, and immunostimulating effects⁵⁰, most of which are low molecular weight substances and have significantly contributed to the development of pharmaceuticals as the beginning of numerous medicines. LPS in herbal extracts supposedly causes this immunostimulating effect because previous LPS screening study revealed that some herbal extracts contain high LPS amounts (> 100 μg/g)³² and previous studies have shown that the oral intake of LPS enhances immunity and effectively prevents and improves various diseases, including cancer, viral infection, atopic dermatitis, diabetes, atherosclerosis, and Alzheimer’s disease^38,51–53. Although there are more than several hundred herbal extracts worldwide and the possibility that the LPS in these herbal extracts playing a role in their functions is high, the LPS amount in them has never been measured or compared among the parts of plants from which they were obtained. Therefore, this study aimed to create a database of LPS levels in herbal extracts by measuring LPS levels in over 400 herbal extract samples stored at the Faculty of Pharmaceutical Sciences, Hokkaido University of Science, and to provide a basis for research to assess the immunostimulatory effects of herbal extracts and LPS’s contribution to these effects.

Table 1 shows the amount of LPS in 414 herbal extracts. LPS concentrations were widely distributed from a few μg/g to several hundred μg/g (Fig. 1). LPS content was shown to be significantly higher in roots (107 samples) than in fruits (69 samples) or seeds (22 samples) in terms of LPS concentration. Of the 414 herbal extracts measured in this study, approximately 100 herbal extracts contained ≥ 10 μg/g of LPS. Twelve of the herbal extracts exhibited very high LPS levels of over 100 μg/g. Comparison among vascular plant parts showed that the overall LPS level in root-derived herbal extracts was high and significantly higher than that in seed- and fruit-derived herbal extracts. Over half (seven) of the 12 high-LPS-content herbal extracts were root-derived. Most vascular plants are symbiotic with soil bacteria in their roots^54–56. Symbiotic bacteria in soil promote plant growth through their involvement in nitrogen fixation, nutrient supply, and disease defense. Such bacteria are called plant growth-promoting rhizospheric microorganisms (PGPR)⁵⁷; among them, bacteria of the genera Pseudomonas, Azospirillum, Bradyrhizobium, and Rhizobium are particularly essential. These bacteria are gram-negative bacteria and, therefore, may contribute to the high-LPS-content in the roots of herbal extracts. Montenegro et al. reported that 519 genera of bacteria are found in Angelica sinensi, a root-derived herbal extract that constitutes Juzen Daihoto, a Chinese herbal medicine known for its immunostimulating properties⁴³. Among them, Rahnella, a gram-negative bacterium found in soil and fresh water, is abundant in Angelica sinensi. It was stipulated that the LPS content in Angelica sinensi is involved in the immunity-enhancing effects of Juzen Daihoto. The LPS content of Angelica sinensi (also called Angelica acutiloba Kitag. in Japan) was also measured in this study and it was shown that it contained 16 μg/g LPS, the 61st highest LPS content among all 414 samples in Table 1 (herb sample no. 202). These results suggest that the LPS amount in the root-derived herbal extract correlates with the number of soil-derived microorganisms that symbiotically coexist with the root-derived microorganisms during growth. These microorganisms are mostly gram-negative bacteria that contain a high LPS amount. On the other hand, the variation within each part group is large, suggesting that the high or low LPS content may not so much dependent on the part of the sample.

The amount of LPS contained in plants is considered to be derived from symbiotic bacteria. Therefore, the type and amount of symbiotic bacteria may vary depending on the origin of the plant, time of collection, variety, and cultivation method. Consequently, it is meaningful to measure multiple samples, but it is difficult to obtain multiple lots of crude drugs because most of them are imported. Therefore, we decided to use the variation in LPS content of one crude drug, brown rice, as a model for the variation in a single crude drug sample. In a previous study, we obtained brown rice from 15 different locations in Japan and measured LPS content in the 10.9 ± 4.3 μg/g range⁵⁸. Although the LPS content of brown rice may not necessarily be universalizable to other crude drugs, we believe that this can be used as a reference value for the degree of variation in LPS content. The range of LPS content in this one sample was relatively stable compared to the range of 0.001–100 μg/g in the LPS content data (Table 1, Fig. 1) obtained for individual crude drugs. Therefore, based on this fact, we conducted the experiment with the belief that the approximate degree of LPS content could be evaluated with a single sample.

In this study, Limulus amebocyte lysate (LAL) test was used to detect LPS in the herbal extracts. However, it has been reported that β-1,3-glucan also reacts with LAL, so, there is a possibility of measuring plant-derived β-1,3-glucan contaminant with ordinary LAL. In this study, this contamination is prevented by using an LAL test kit containing a carboxymethylated curdlan which has reported act as a blocker of β-1,3-glucan mediated coagulation pathway⁵⁹. Therefore, the limulus activity detected in this study were specific to LPS.

The macrophage-activating ability of LPS is a fundamental LPS action³⁴. Therefore, the macrophage activation potential of herbal extracts by phagocytosis and NO production was assessed using macrophage-like RAW 264.7 cells. RAW246.7 cells transduce LPS signaling via TLR4⁶⁰. In addition, many mammalian innate immune system cells, including humans, express TLR4⁶¹. Therefore, even though this study used mouse macrophage cells as a representative model, it is safe to assume that LPS contained in crude drugs is functional for mammals in general, including humans. However, further research is needed to determine the effects of LPS in humans, especially when administered orally. Twelve samples containing particularly high amounts of LPS (100 μg/g) were examined using these methods. The results showed that herbal extracts increased the phagocytosis capability of RAW 264.7 cells (Fig. 2). The NO production by RAW 264.7 cells caused by these samples was found to be higher, similar, or lower than purified LPSp, depending on the 12 herbal extracts (Fig. 3). The LPS itself in the group that exhibited higher activity may display high macrophage activation. However, it is speculated that a synergistic effect with macrophage activators, such as bacterial-derived nucleic acids, peptidoglycans, and flagellin, may be observed. Conversely, those that exhibited weaker activity than LPSp derived from Enterobacteriaceae may be because of the nature of the symbiotic gram-negative bacteria, as some LPSs, such as Bacteroides, are weak in biological activity, which depends on their lipid A structure^62,63. Additionally, NO production was significantly (> 70%) reduced in all RAW 264.7 cells stimulated with 12 herbal extracts when polymyxin B, an LPS inhibitor, was added (Fig. 4). These results suggest that LPS is responsible for most of the macrophage activation potential of herbal extracts. However, the strength of the macrophage-activating ability of the herbal extracts is not proportional to the amount of LPS contained and may significantly differ among various symbiotic bacteria. Therefore, in studying the innate immune activation potential of herbal extracts, it is necessary to assess and clarify their unique qualities.

Herbal extracts are often prescribed in daily doses of 1–10 g^64,65. Of the 414 herbal extracts for which LPS levels were measured in this study, 98 contained over 10 µg/g LPS, and oral intake of LPS increased the phagocytic activity of abdominal macrophages in mice at 10 µg/kg BW for 7 days³⁴, induced increase in capillary vascularity at 10 µg/kg BW in human randomized control trial studies⁶⁶, and in fish, 5–20 μg/kg BW increased the ability to prevent infection⁶⁷. Based on these studies, 10 μg/kg BW of LPS can activate innate immunity, which is 500 μg/day for a 50 kg human. Therefore, consuming a daily dose of herbal extracts may mean taking in an effective amount of LPS, meaning that LPS may contribute to the medicinal effects of the herbal extracts. Juzen Daiho-to, a combination of herbal extracts, reportedly has preventive and ameliorative effects against diabetes and cancer partly because LPS is one of its ingredients^68,69. The 414 herbal extract samples measured in this study are much greater than the 157 listed in the Japanese Pharmacopoeia. These should be sufficient populations for primary screening based on the efficacy of oral LPS intake over immune functions and the activation of immune cells using macrophages and other cells in herbal extracts. However, because the LPS content of plants is obtained from the symbiotic gram-negative bacterial population and may differ greatly depending on the time of collection, variety, cultivation method, etc., the LPS content of the samples to be studied should be analyzed with caution on a sample-by-sample basis.

Methods

Sample preparation

All dried samples were purchased from Tochimoto Tenkaido Co., Ltd. (Osaka, Japan). The dried samples were pulverized, and 100 mg powdered samples were extracted in 1 ml distilled water for 20 min at 90 °C. Subsequently, the samples were sonicated for 20 min and vortexed for two minutes to extract LPS. Next, the supernatants were obtained after centrifugation at 830 × g for 15 min. All methods involving the dried samples were carried out in accordance with relevant guidelines⁷⁰.

Measurement of the LPS contents of herbal extracts

The LPS concentration in the samples were assayed using the kinetic–turbidimetric method. All samples were diluted 10,000-fold using pyrogen-free distilled water. Sample supernatants (0.2 ml) were added to LAL-ES in a glass tube (Limulus ES-II single test; Wako Pure Chemical Industries Ltd., Osaka, Japan). After a few seconds of votexing, the gelation time was measured using a Toxinometer ET-6000 (Wako Pure Chemical Industries Ltd.), and the specific activity was calculated using an LS Toximaster (Wako Pure Chemical Industries Ltd.), a data acquisition program for the Toxinometer.

The LAL test kits of Wako contain carboxymethylated curdlan in freeze-dried reagents, which stops β-d-glucans from triggering an interference in the test. Therefore, this test kit used in this study is specific to LPS⁵⁹.

Phagocytosis assay

Phagocytic activity was measured using flow cytometry as previously described with minor modifications⁷¹. Briefly, the mouse macrophage/monocyte cell line RAW 264.7 cells (obtained from TIB-71; ATCC, Manassas, VA, USA) were treated for 18 h with extracts in a 48-well plate. The extract concentrations were measured so that the LPS content was 100 ng/ml. Next, fluorescent latex beads (Fluoresbrite^® YG Microspheres 1.0 μm; Polysciences, Warrington, PA) at a cell: bead ratio of 1:10 were added and incubated for one hour. Cells were washed to eliminate non-internalized particles and detached from the well plate with 0.25% trypsin treatment (Life Technologies, Carlsbad, CA, USA). The phagocytosis rate of the cells was measured using a Beckman Coulter Gallios flow cytometer and Kaluza software (Beckman Coulter, Indianapolis, IN).

Nitric oxide (NO) production by murine macrophages

In a 48-well plate, cells from the mouse macrophage/monocyte cell line RAW 264.7 were plated at 8 × 105 cells/ml and treated with herbal extracts. The added extract concentrations were measured, so that the LPS content was 1, 10, and 100 ng/ml. The plate was incubated at 37 °C and 5% CO₂. After 24-h incubation with extracts, the supernatants were collected, and the concentrations of nitrite (NO²⁻) released into the culture media were measured using Griess reagent. In addition, 100 μl Griess reagent was added to 100 μl diluted culture media in the wells of microtiter plates. After incubation at room temperature for ten minutes, absorbance at 570 nm was determined using an automated microplate reader (BIO-RAD, Hercules, CA, USA). The NO assay was conducted in duplicate. To determine the percentage of NO produced by the LPS in the herbal extracts, the concentrations of the extracts were measured, so that the LPS content was 10 ng/ml, and polymyxin B (Sigma-Aldrich, St. Louis, MO, USA) was added to each culture at a final concentration of 10 μg/ml.

Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical analyses (Steel–Dwass test and Pearsons’ correlation) were performed using the JMP statistical software, version 17. 0. 0 (SAS Institute Inc., Cary, NC, USA). Statistical differences between multiple groups in the box-and-whisker plot were calculated using the Steel–Dwass test. A p-value < 0.05 was considered statistically significant. The line equation and its R² value in Table 3 were performed using Microsoft Excel.

Author contributions

Conceptualization, K.W., H.I. and G.S.; methodology, H.I.; software, M.O. and M.Y.; validation, M.Y.; formal analysis, M.Y.; investigation, M.Y.; resources, K.W.; data curation, M.Y., T.I., Y.F.; writing—original draft preparation, V.T. and M.O.; writing—review and editing, V.T., M.O., T.I., K.W., H.I. and G.S.; visualization, M.Y.; supervision, H.I. and G.S. All authors have read and agreed to the published version of the manuscript.

Data availability

All data generated or analyzed during this study are included in this published article.

Competing interests

The authors declare no competing interests.