Health benefits of 4,4-dimethyl phytosterols: an exploration beyond 4-desmethyl phytosterols

Abstract

4,4-Dimethyl phytosterols possess two methyl groups at the carbon-4 atom of the aliphatic A-ring. The methyl groups are crucial for the molecular recognition of endogenous and exogenous bioactive compounds. Phytosterols have received worldwide attention owing to their recognized health benefits. However, 4,4-dimethyl phytosterols are less appreciated. Recent research studies revealed that 4,4-dimethyl phytosterols exert numerous beneficial effects on disease prevention, and are particularly involved in the endogenous cannabinoid system (ECS). The purpose of this review is to summarize and highlight the currently available information regarding the structures and sources of 4,4-dimethyl phytosterols, and to provide detailed preclinical studies performed to evaluate their potential for treating various diseases. Future research on 4,4-dimethyl phytosterols is warranted to confirm their relationship with the ECS, and to elucidate the mechanism directly toward clinical trials.

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1. Introduction

Plant sterols share a fused polycyclic structure and differ in the number or position of unsaturated bonds, and the length of the substitutents and side chains. Based on the location of methyl groups at the carbon-4 position, phytosterols are classified into 4,4-dimethyl phytosterols, 4-monomethyl phytosterols, and 4-desmethyl phytosterols.¹ More than 250 unique phytosterols have been identified in recent years.² The 4,4-dimethyl phytosterols are quantitatively minor in nature, but are typically found in plants and vegetable oils. Some belonging to the triterpene family also contain two methyl substituents, thus falling into the group of 4,4-dimethyl phytosterols that includes lupeol, α-amyrin, β-amyrin, cycloartanol, taraxerol, etc.^3,4 The 4-desmethyl phytosterols have received worldwide attention due to their efficient cholesterol-lowering activity. The most common 4-desmethyl phytosterols are β-sitosterol, stigmasterol, and campesterol. Many reviews address the progress of the structure and variation of 4-desmethyl phytosterols,⁵ as well as the metabolism and function in vivo.^6–8 However, a comprehensive review about the benefits of 4,4-dimethyl phytosterols has not appeared.

The 4,4-dimethyl phytosterols are naturally occurring bioactive compounds, and resemble 4-desmethyl phytosterols in many functions.^9,10 According to the World Health Organization (WHO), noncommunicable diseases (NCDs) play a key role in the global mortality. Previous studies demonstrated that the 4,4-dimethyl phytosterols can mitigate NCDs and associated disorders. Examples are obesity suppressed by α,β-amyrin,^9,11 diabetes prevented by taraxerol,^12,13 as well as hyperlipidemia ameliorated by lupeol, 24-methylene cycloartanol, and cycloartenol in animals or humans.¹⁴ In addition to regulating lipid metabolism, 4,4-dimethyl phytosterols also exhibited anti-inflammatory, anticancer, and antinociceptive activities.

Though diverse factors account for the death rate worldwide, there seems to be a relationship between 4,4-dimethyl phytosterol intake and the death rate from NCDs (Fig. 1). As shown in Fig. 1, inside the black dotted box, the regions with a higher intake of 4,4-dimethyl phytosterols seem to have lower mortality from NCDs, such as North America, Mediterranean, East Asia, and Australia. In-depth studies ascertained the linkages between the 4,4-dimethyl phytosterols and the endogenous cannabinoid system (ECS), whose dysregulation could cause various NCDs.¹⁸ This review highlights the structures and sources of naturally occurring 4,4-dimethyl phytosterols, and provides a detailed account of the preclinical studies performed to evaluate the effectiveness of 4,4-dimethyl phytosterols as functional compounds for disease treatments.

Fig. 1 Comparison of death rates due to NCDs (A) and 4,4-dimethyl phytosterol intake in the world (B). Data of the age-standardized death rate (2015) due to NCDs are from the World Health Organization (WHO), produced by Information Evidence and Research (IER).¹⁵ Based on the 4,4-dimethyl phytosterols being mainly consumed from vegetable oils, the 4,4-dimethyl phytosterol intake is calculated from their content in vegetable oils. The consumption data of different vegetable oils in 2014 worldwide was reported by the Food and Agriculture Organization (FAO).¹⁶ The content of 4,4-dimethyl phytosterols in each kind of vegetable oil is obtained from Bailey's Industrial Oil and Fat Products (sixth edition).¹⁷

2. Diversity of natural 4,4-dimethyl phytosterols

2.1. Structure of 4,4-dimethyl phytosterols

The key characteristics and structural peculiarities of 4,4-dimethyl phytosterols are shown in Fig. 2, which includes a 3β-hydroxyl (3β-OH) group and a side chain with variable numbers of carbons.

Fig. 2 Summary of the structure–compound relationship of the major 4,4-dimethyl phytosterols. The 4,4-dimethyl phytosterols typically possess two methyl groups at the carbon-4 atom position of the aliphatic A-ring. Due to the structural differences in the aliphatic rings, 4,4-dimethyl phytosterols are further classified into a common type (1), a cycloartanol type (2), and a triterpene type (3). The dotted line and round spot in the ring indicated the linkage positions between the two parts.

The distinctive feature of 4,4-dimethyl sterols is the existence of two methyl substituents at the carbon-4 position in the aliphatic fragment. Similar to 4-desmethyl phytosterols, 4,4-dimethyl phytosterols also contain a 3β-OH group and one or several unsaturated bonds in the ring or the side chain. Esterification compounds of 4,4-dimethyl phytosterols are formed by coupling the 3β-OH group with free fatty acids, carbohydrates, or phenolic acid, thus forming the corresponding esters, such as oryzanol.

Until now, many different types of 4,4-dimethyl phytosterols have been identified, which vary mostly in their substitutents at carbon-9 and carbon-17 positions, and their hydrophobic side chains (Fig. 2). The chemical difference among the common type of 4,4-dimethyl phytosterols is attributed to the numbers of carbon atoms and the presence of unsaturated bonds in the side chain linked with carbon-17, such as lanosterol (1a), euphol (1b), tirucallol (1c), etc. Considering the structural distinction of unsaturated bonds, the substituents at the carbon-24 position, and the stereochemical properties, 4,4-dimethyl phytosterols are further divided by the cyclization at the carbon-9 position or variations of the divergent ring, thus forming the cycloartenol type of 4,4-dimethyl phytosterols (2a, 2b, 2c, 2d, 2e, 2f, etc.) and the triterpene type of 4,4-dimethyl phytosterols (3a, 3b, 3c, 3d, etc.).

2.2. Sources of 4,4-dimethyl phytosterols

Plants synthesize a more diverse range of 4,4-dimethyl phytosterols than animals or microorganisms. Considerable amounts of α-amyrin, β-amyrin, taraxerol and lupeol have been reported to be present in the plant kingdom. Some of the above 4,4-dimethyl phytosterols have been reported previously.^19,20 However, in the current review, other sources of the 4,4-dimethyl phytosterols are further summarized and compared (Table 1). α,β-Amyrin mainly existed in plant resins of the Bursera and Protium species in the Burseraceae family.²⁰ Lupeol is primarily distributed in vegetables, fruits and medicinal plants.²¹ Compared to α,β-amyrin, lupeol is commercially available, relatively low cost, and always of high yield. Moreover, many medicinal plants are also rich in taraxerol. A polar extract of Clitoria ternatea contains taraxerol at a content of 12.4 mg g⁻¹.²² High contents of taraxerol can be extracted from Vaccinium oldhamii (22 μg g⁻¹ dry twigs) and Eugenia umbelliflora (35 μg g⁻¹ dry fruits).^23,24 As for euphol, the fresh leaf of Euphorbia tirucalli could yield approximately 4.06 mg g⁻¹ of euphol.²⁵

Table 1 Sources of naturally occurring 4,4-dimethyl phytosterols

Sources	4,4-Dimethyl phytosterols	Ref.
a Represent the predominant compound in the corresponding source.
Plant source
Canavalia ensiformis seeds	Lupeol^a, β-amyrin	34
Rhizophora racemose leaves	Lupeol^a, α-amyrin, β-amyrin	37
Brassica oleracea leaves	Lupeol^a, α-amyrin, β-amyrin,	38
Bursera cuneate resin	Lupeol^a, α-amyrin, β-amyrin	39
Adenophora triphylla roots	Lupeol^a, taraxerol	40
Vitex trifolia fruits	Lupeol, α-amyrin, taraxerol	41
Ricinus communis leaves	Lupeol^a, α-amyrin, β-amyrin	42
Coccoloba uvifera leaves	Lupeol^a, α-amyrin, β-amyrin	43
Betula alleghaniensis leaves	α-Amyrin^a, β-amyrin	44
Canarium luzonicum resin	α-Amyrin^a, β-amyrin	45
Capsicum annuum skin	α-Amyrin^a, β-amyrin, taraxerol	46
Protium heptaphyllum resin	α-Amyrin^a, β-amyrin	47
Protium burseraceae resin	α-Amyrin^a, β-amyrin	48
Hieracium pilosella roots	α-Amyrin^a, β-amyrin, taraxerol	49
Bursera copallifera resin	α-Amyrin^a, lupeol	50
Zea mays leaves	β-Amyrin^a, α-amyrin	51
Nelumbo nucifera pollen	β-Amyrin^a, α-amyrin	52
Vaccinium myrtillus leaves	β-Amyrin, 24-methylene cycloartanol, lupeol	53
Leea macrophylla leaves	β-Amyrin^a, lupeol	54
Euphorbia kansui roots	Euphol^a, β-amyrin, α-amyrin	55
Synadenium grantii barks	Euphol^a, lanosterol	56
Mangifera indica barks	Taraxerol^a, β-amyrin, α-amyrin	57
Taraxacum officinale aerial	Taraxerol^a, lupeol, β-amyrin	58
Dioscorea tenuipes leaves	Cycloartenol^a, α-amyrin, 24-methylene cycloartanol	59
Vegetable oil source
Carrot root oil	24-Methylene cycloartanol^a, cycloartanol, β-amyrin	60
Rice bran oil	24-Methylene cycloartanol^a, cycloartenol, cycloartanol	61
Olive oil	24-Methylene cycloartanol^a, cycloartenol	62
Wheat germ oil	24-Methylene cycloartanol^a, β-amyrin, cycloartenol	63
Eggplant seed oil	Cycloartanol^a, cycloartenol	64
Camellia seed oil	Cycloartanol^a, cycloartenol, 24-methylene cycloartanol	65
Sea buckthorn seed oil	Lanosterol^a, β-amyrin, cycloartenol	66

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In addition to plant sources, dietary phytosterols are mostly derived from vegetable oils, cereals, nuts, and fruits. Vegetable oil is predominantly used and is estimated to represent 40% of the total intake in China,²⁶ and about 26% in the Netherlands and Sweden.²⁷ The 4,4-dimethyl phytosterols, mostly 24-methylene cycloartanol and cycloartanol, are presented as free or esterified forms in various vegetable oils. Rice bran oil has already been recognized for its high content of 24-methylene cycloartanol (4.94 mg g⁻¹) and cycloartenol (4.82 mg g⁻¹).¹⁷ Soybean oil is also rich in cycloartenol (1.68 mg g⁻¹), while the 24-methylene cycloartanol content in sesame seed oil reaches about 1.07 mg g⁻¹.²⁸ Moreover, the 24-methylene cycloartanol has been reported at a higher level in olive oil as compared to hazelnut oil.²⁹ The quantitative analysis of 4,4-dimethyl phytosterols indicated a significant difference in the content of cycloartenol between different edible oils.³⁰ Thus, the 4,4-dimethyl phytosterols can be utilized as a possible marker in the detection of oil adulteration. However, 4,4-dimethyl phytosterols from microbial and animal sources are limited due to their different biosynthetic pathways. Previous studies adopted bioengineering techniques to clone synthase genes of 4,4-dimethyl phytosterols from plants and express on microorganisms to produce the targeted 4,4-dimethyl phytosterols.^31,32 Notably, the fat of sheep wool can be developed as a suitable source of lanosterol. The fresh wool contained about 20 mg g⁻¹ of the total sterol, and lanosterol was about 1.84 mg g⁻¹.³³

Notably, the neutral lipid extracted from the seeds of Canavalia ensiformis is a promising source of lupeol. The triterpene alcohol fraction was mainly composed of lupeol (96.3%), with the minor constituent being β-amyrin.³⁴ Additionally, the unsaponifiable matter of shea butter yielded the 4,4-dimethyl phytosterols as the predominant constituent, which consisted of α-amyrin (26.5%), lupeol (21.7%), and β-amyrin (10.2%).^35,36 Overall, the major sources of naturally occurring 4,4-dimethyl phytosterols are presented in Table 1.

3. Health benefits of 4,4-dimethyl phytosterols

3.1. Anti-inflammatory activity

The inflammatory process would raise the level of cytokines such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-1β, IL-6 and IL-17A in serum, necrotic cells or damaged tissues.⁶⁷ The 4,4-dimethyl phytosterols utilized as anti-inflammatory agents have been widely reported (Table 2).

Table 2 In vivo studies of the anti-inflammatory activity of 4,4-dimethyl phytosterols

Compound	Inflammation	Experimental design	Administration	Dose	Outcomes	Ref.
Not specified, N.S.; intraperitoneal injection, i.p.; tumor necrosis factor-α, TNF-α; interleukin-6, IL-6; myeloperoxidase, MPO; thiobarbituric acid-reactive substances, TBARS; dimethylnitrosamine, DMN; tissue inhibitor of metalloproteinase-1, TIMP-1; complete Freund's adjuvant, CFA; interleukin-1β, IL-1β; transcription factor-κB, NF-κB; keratinocyte-derived chemokine, KC; cyclic AMP response element binding protein, CREB; cyclooxygenase-2, COX-2; trinitrobenzene sulfonic acid, TNBS; interleukin-10, IL-10; prostaglandin E2, PGE2; 12-O-tetradecanoyl-phorbol-13-acetate, TPA; dextran sodium sulfate, DSS; inducible nitric oxide synthase, iNOS; lipopolysaccharide, LPS; interferon-γ, IFN-γ; myelin oligodendrocyte glycoprotein, MOG35-55; monocyte chemoattractant protein-1, MCP-1; monocyte chemoattractant protein-2, MIP-2; nitric oxide, NO; and phospholipase A2, PLA2.
Lupeol	Ear oedema	Male Swiss mice were topically treated with TPA (2.5 μg) to the right ear	Topical	0.5 and 1.0 mg per ear	Nitrite↓, PGE2↓, TNF-α↓, IL-1β↓	69
Tirucallol	Ear oedema	Male Swiss mice were topically treated with TPA (2.5 μg) to the right ear	Topical	0.25, 0.5, and 1.0 mg per ear	Weight edema↓, NO↓, PGE2↓, iNOS↓	71
α,β-Amyrin	Periodontitis	Nylon ligature was placed around the maxillary right tooth of male Wistar rats	Oral	5 and 10 mg kg^−1	TNF-α↓, MPO↓, TBARS↓	79
β-Amyrin	Hepatic fibrosis	Male Wistar rats were injected (i.p.) with DMN in saline (10 μL kg^−1)	Oral	30 mg kg^−1	TNF-α↓, caspase-3↓, TIMP-1↓	80
Lupeol	Allergic airway inflammation	Male BALB/c mice were exposed in a box with aerosolized ovalbumin (1%)	Oral	60 mg kg^−1	IL-4↓, IL-5↓, IL-13↓	83
α,β-Amyrin	Colitis	Male Swiss mice were injected (i.p.) with TNBS (25 mg mL^−1, 100 μL)	Intraperitoneal	0.3, 1.0, and 3 mg kg^−1	MPO↓, IL-10↓, IL-1β↓, COX-2↓, NF-κB↓, CREB↓	84
Taraxerol	Paw oedema	Male Wistar rats were injected (i.p.) with carrageenan (1%, 0.1 mL)	Intraperitoneal	5 and 10 mg kg^−1	IL-1β↓, IL-6↓, IL-12↓, TNF-α↓, NF-κB↓, COX-2↓, iNOS↓	85
α,β-Amyrin	Pancreatitis	Male Wistar rats were injected (i.p.) with L-arginine (2.5 g kg^−1)	Oral	10, 30, and 100 mg kg^−1	TNF-α↓, IL-6↓, MPO↓, TBARS↓, nitrate/nitrite↓	86
α,β-Amyrin	Pancreatitis	Male Swiss mice were injected (i.p.) with cerulean (50 μg kg^−1)	Oral	10, 30, and 100 mg kg^−1	TNF-α↓, IL-6↓, MPO↓, TBARS↓	91
α-Amyrin	Skin inflammation	Male Swiss mice were topically treated with TPA (2.5 μg per ear)	Topical	0.1, 0.2, and 1.0 mg per ear	NF-κB↓, PGE2↓, COX-2↓	92
α-Amyrin	Ear oedema	Male Swiss mice were topically treated with TPA (2.5 μg per ear) to the right ear	Topical	0.1, 0.5, and 1.0 mg per ear	MPO↓, IL-1β↓	93
Lupeol	Colitis	Male Swiss mice were treated with 2% DSS in drinking water	Intraperitoneal	40 mg kg^−1	MPO↓, TNF-α↓, IL-2↓, IL-6↓, NF-κB↓	94
Lupeol	Colitis	Male C57BL/6 mice were treated with 4% DSS in drinking water	Oral	50 mg kg^−1	IL-12↓, IL-6↓, IL-1β↓, TNF-α↓, IL-10↑	95
Lupeol	Neurogenic inflammation	Male C57BL/6 mice were injected (i.p.) with LPS (250 μg kg^−1 day^−1)	Oral	50 mg kg^−1	TNF-α↓, iNOS↓, IL-1β↓	96
Lupeol	Pleuritis	Male Swiss mice were injected (i.p.) with carrageenan (1%, 0.1 mL)	Oral	25, 50, 100, and 200 mg kg^−1	IL-2↓, IFN-γ↓, TNF-α↓	97
Euphol	Colitis	Male Swiss CD-1 mice were treated with 3% DSS or TNBS (15 mg mL^−1, 100 μL)	Oral	3, 10, and 30 mg kg^−1	MCP-1↓, CXCL/KC↓, IL-1β↓, MIP-2↓, TNF-α↓, IL-6↓, IFN-γ↓, IL-10↑	98
Taraxerol	N. S.	Male Wistar rats were fed with HFD and STZ (i.p. 35 mg kg^−1)	Oral	20 mg kg^−1	MCP-1↓, IL-1β↓, IL-6↓, TNF-α↓, NF-κB↓	99
Cycloartenol	Hind-paw oedema	Male rats were injected (i.p.) with carrageenan (0.1 mL, 1%) to the right hind paws	Oral	10, 20, and 40 mg kg^−1	PLA2↓, leucocyte migration↓	100
Cycloartenol, 24-methylene cycloartanol	Ear oedema	Female ICR mice were topically treated with TPA (1.0 μg) to the right ear	Topical	0.2 and 0.3 mg per ear	Ear thickness↓	101
Euphol	Skin inflammation	Male Swiss CD-1 mice were topically treated with TPA (2.5 μg) to the right ear	Topical	50 and 100 μg per ear	MPO↓, NAG↓, COX-2↓, CXCL/KC↓, MIP-2↓	109

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Gupta et al. firstly found that the 4,4-dimethyl phytosterols exerted an anti-inflammatory activity against arthritis and oedema.⁶⁸ Since then, 4,4-dimethyl phytosterols have been extensively demonstrated to exhibit anti-inflammatory activities. Several studies conducted by Fernandez et al. indicated that the 4,4-dimethyl phytosterols were effective in alleviating the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced ear oedema.^69–71 Lupeol pretreatment decreased the myeloperoxidase (MPO) activity, the cell infiltration in damaged tissues, and prostaglandin E2 (PGE2) in macrophagocytes.⁶⁹ The related mechanism was similar to that of indomethacin, which is an effective inhibitor of cyclooxygenase (COX).⁷⁰ Tirucallol also prevented the inflammation via a mechanism related to the reduction of nitric oxide (NO) and inducible nitric oxide synthase (iNOS).⁷¹ Additionally, taraxerol and β-amyrin exhibited a similar anti-inflammation effect on ear oedema. However, α-amyrin and 24-methylene cycloartanol exhibited higher inhibition capacity.⁷²

The anti-inflammation benefits of 4,4-dimethyl phytosterols were associated with the modulation of the immune system and reduction of cytokines in an arthritis model.^73,74 Lupeol could decrease the collagen, thereby increasing the activities of lysosomal enzymes.⁷⁵ Though the detailed mechanism was not given, the CD4⁺ T cell-mediated autoimmune response is critical in the prevention of arthritis.⁷⁶ Eliminating CD4⁺ T cells would contribute to the therapy of arthritis.⁷⁷ Therefore, a subsequent study focused on CD4⁺ T cells indicated that lupeol could modulate the phagocytic function of T-lymphocytes and macrophages, thus inhibiting the generation of pro-inflammatory cytokines.⁷⁸ The above results illustrated that lupeol was effective at suppressing T cells, which is suggestive of future utilization. Moreover, α,β-amyrin modulated the periodontal inflammation through inhibiting neutrophil infiltration and oxidative stress.⁷⁹ However, β-amyrin treatment alone attenuated the altered oxidative stress, TNF-α levels, and protein expression.⁸⁰ β-Amyrin acetate was found to exert a stronger anti-inflammatory activity than α-amyrin acetate.^68,81 The above studies indicated that β-amyrin might exhibit a higher anti-inflammatory activity. However, another study showed an opposite result on the inhibition of TPA-induced inflammation.⁷² The inconsistent results can be attributed to the different inflammatory models utilized.

The 4,4-dimethyl phytosterols were also compared with some anti-inflammatory compounds (e.g. α-mangostin, dexamethasone, and methylprednisolone). The comparison of lupeol and α-mangostin demonstrated that lupeol exhibited 57.14% inhibition, while α-mangostin at a similar dose showed only 38.70% inhibition.⁸² Similarly, in allergic airway inflammation, lupeol was observed to be as effective as dexamethasone, and significantly decreased the generation of mucus and pro-inflammatory cytokines in the lungs.⁸³ Another study also showed a similar result in reversing the colitis with α,β-amyrin, which indicated that the inhibition of transcription factor-κB (NF-κB) and the cyclic AMP response element binding protein (CREB) was the possible mechanism.⁸⁴ However, only when treated with a higher dose at 10 mg kg⁻¹, the inhibitory activity of taraxerol was comparable to that of dexamethasone (0.5 mg kg⁻¹).⁸⁵ Based on these results, the α,β-amyrin might exhibit a stronger activity than taraxerol, which was also confirmed in the study conducted by Akihisa et al.⁷² Moreover, in a pancreatitis model, the activity of α,β-amyrin was equivalent to that of methylprednisolone,⁸⁶ and the esterified derivatives of 4,4-dimethyl phytosterols (acetate, palmitate and linoleate) have been reported to display higher anti-inflammatory activities.^87–89 Esterification can enhance the activity by increasing the diffusion, absorption, and retention capacity, which might explain the higher anti-inflammatory activities of the esterified derivatives.

The 24-methylene cycloartanol and α-amyrin exerted the strongest activity with an IC₅₀ dose of 0.2 mg per ear.⁷² Similarly, Yasukawa et al. indicated that the 24-methylene cycloartanol exhibited the highest suppression activity on tumor growth.⁹⁰ In brief, these results confirmed the health benefits of 4,4-dimethyl phytosterols in various inflammatory models, suggesting that the supplementation with 4,4-dimethyl phytosterols is a potential approach to treat inflammation. However, it should be mentioned that the inconsistent results still exist, and more studies are thus needed to explore the anti-inflammatory difference between different 4,4-dimethyl phytosterols, and to determine which kind of 4,4-dimethyl phytosterol can treat the inflammation in the target.

3.2. Anticancer activity

Topical application of 4,4-dimethyl phytosterols modulated the pathways involved in growth, survival and apoptosis in several models of skin cancer.^102,103 Considering that the NF-κB, phosphatidyl inositol 3-kinase (PI3K)/Akt pathway plays a critical role in cancer promotion, Saleem et al. proved that lupeol showed anti-skin cancer ability by inhibiting the activation of NF-κB and PI3K/Akt.¹⁰³ Moreover, cycloartenol is another effective 4,4-dimethyl phytosterol for treating skin cancer. Cycloartenol could suppress the benzoyl peroxide and ultraviolet-B radiation-induced cellular oxidative stress, and reverse the induction of cutaneous ornithine decarboxylase (ODC) activity.¹⁰⁴ Euphol could prevent the tumor-promoting effect of TPA initiated with 7,12-dimethylbenz[a]anthracene (DMBA) by reducing the levels of keratinocyte derived chemokine (CXCL1/KC) and macrophage inflammatory protein-2 (MIP-2), which are related to the expression of protein kinase C.^90,102 Several anti-cancer effects of 4,4-dimethyl phytosterols were compared.¹⁰⁵ Taraxerol exhibited the most potent anti-tumor activity as compared to α-amyrin, β-amyrin, and lupeol. A surprising observation is that the anticancer activity of taraxerol acetate was weaker than that of taraxerol,¹⁰⁵ which is different from the anti-inflammatory effect and remains to be reconfirmed.

In addition to skin cancer, 4,4-dimethyl phytosterols exhibited anticancer effects in many other cancers, including oral cancer, bladder cancer, liver cancer, colon cancer, etc. Ovadje et al. investigated several 4,4-dimethyl phytosterols for their effects on colon cancer.¹⁰⁶ Compared to β-amyrin and lupeol, α-amyrin was more effective at decreasing the viability of colon cancer cells. No synergistic interactions between different 4,4-dimethyl phytosterols were observed. Lanosterol inhibited the formation of colonic aberrant crypt foci (ACF) and crypt multiplicity.¹⁰⁷ Prasad et al. found that lupeol significantly decreased cyclooxygenase-2 (COX-2) in the bladder and nuclear matrix protein 22 (NMP22) in urine.¹⁰⁸ Lupeol was also effective at inhibiting stress-induced liver injury through moderating apoptosis factors. Meanwhile, lupeol could decrease the peroxidation levels and restore the antioxidant enzymes in favor of the excretion of carcinogenic metabolites, thus inhibiting tumor growth.¹⁰⁸ Hong et al. investigated the anticancer effect of taraxerol ester in U87 human glioblastoma cells. Taraxerol acetate significantly reduced the tumor weight and size in a xenograft model.¹⁰⁹ Therefore, the above results proved that the 4,4-dimethyl phytosterols are effective anticancer agents.

3.3. Anti-Alzheimer's and anti-Parkinsonian activities

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most prevalent and severe neurological diseases which are basically disorders in the functions of nervous systems.¹¹⁰ No effective drug as yet could provide complete therapy for PD and AD. The 4,4-dimethyl phytosterols have been reported to exert biological functions against AD and PD.

The inhibition of acetylcholinesterase (AChE) was believed to improve the condition of AD patients.¹¹¹ The activity of AChE could be inhibited by β-amyrin and taraxerol, but not α-amyrin.^112,113 However, the expression level of memory-related signal transduction proteins was significantly enhanced by both α-amyrin and β-amyrin in the hippocampus, which suggested that α-amyrin and β-amyrin ameliorated the cognition disorders through the activation of extracellular signal-regulated kinase 1/2 (ERK) and glycogen synthase kinase-3β (GSK-3β), as the ERK inhibitor (U0126) blocked this effect.¹¹⁴ Molecular docking studies revealed that taraxerol had a high affinity for fibrils and β-amyloid peptides, but low toxicity, and confirmed the ability of taraxerol in the inhibition of AChE activity.^113,115 A recent in vivo study further proved the effects of taraxerol on the AChE activity and memory deficits. Taraxerol pretreatment significantly improved memory impairment in the hippocampus induced by scopolamine and streptozotocin.¹¹² The mechanism was related to the activation of the receptor system of AChE.

Additionally, β-site amyloid cleaving enzyme 1 (BACE1) was also involved in the rate-limiting step in the pathogenesis of AD, which motivated researchers to target BACE1 for AD prevention.¹¹⁶ Lupeol was confirmed via enzymatic evaluation and molecular dynamics simulations, holding therapeutic potential for AD treatment through the inhibition of BACE1.^117,118 Notably, lupeol produced higher flexibilities of the protein and local fluctuations in the active site of BACE1, which further implied that the binding activity of lupeol is stronger than that of ursolic acid.¹¹⁷ Recently, lupeol treatment has significantly decreased the diabetes-related disorders of AD, such as cognitive impairment, anxiety and fear-associated behaviors.¹¹⁹ In addition to AD, the relocation of lanosterol synthase from the endoplasmic reticulum to mitochondria, suggesting that lanosterol exhibited anti-Parkinsonian activity probably by protecting the dopaminergic neurons from death.¹²⁰ Moreover, the anti-Parkinsonian mechanism of β-amyrin may be related to autophagy involving LGG-1.¹²¹ Autophagy has been reported to be a therapeutic approach to prevent PD owing to the elimination of the misfolded proteins and injured organelles.¹²² In the process of autophagosome formation, LGG-1 exited for structural maintenance; hence it is utilized as a potential marker.¹²³ It was found that the β-amyrin participated in the LGG-1 related autophagy pathway through improving the LGG-1 expression and the amounts of localized LGG-1 puncta.¹²¹ Furthermore, β-amyrin exhibited a protective effect on dopaminergic neurons by reducing cell damage, and α-synuclein aggregation, which was also involved in the symptoms of PD.¹²⁴

It is noteworthy that the previous studies have linked sterol-mediated cholesterol metabolism to neurological diseases. Low levels of LDL-C were usually associated with a higher occurrence of AD and PD.¹²⁵ Thus, the anti-Alzheimer's and anti-Parkinsonian activities of 4,4-dimethy sterols may also be associated with their ability in regulating cholesterol metabolism. Taken together, these results confirmed the effect of anti-neurological disease and indicated that 4,4-dimethyl phytosterols had potential for therapeutic applications in memory impairment.

3.4. Anti-hyperglycaemic and anti-hypolipidemic activities

Hyperglycemia and hyperlipidemia are often present with chronic inflammation and obesity. The 4,4-dimethyl phytosterols exert anti-hyperglycemic and anti-hypolipidemic activities in obesity related metabolic disorders.

Santos et al. investigated the benefits of α,β-amyrin in high fat diet (HFD)-induced obesity.¹²⁶ The α,β-amyrin significantly reduced the content of total triacylglycerol (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and very low-density lipoprotein (VLDL), while elevating the level of high-density lipoprotein cholesterol (HDL-C) in serum. They further indicated that the beneficial activity of α,β-amyrin involved the protection of beta-cells. Subsequent research proposed a possible mechanism through investigating the effect of α,β-amyrin on the contractile and relaxant vascular responses. Treatment with α,β-amyrin prevented body weight gain, insulin resistance, dyslipidemia, and the endothelial dysfunction, probably through a restoration of the guanylate cyclase-K⁺ channel pathway.¹²⁷ Carvalho et al. found that α,β-amyrin ameliorated HFD-induced obesity via regulating the enzymatic, hormonal, and inflammatory responses, and decreased the adipocyte area, visceral adiposity, and the blood lipase activity.⁹ These results indicated the potential of α,β-amyrin in the therapy of obesity and related disorders.

PPAR-α is critical for the regulation of lipids and glucose metabolism, acting as a sensor of the fatty acid load.¹²⁸ Elevation in the circulating fatty acids or the metabolites results in the activation of PPAR-α, which, in turn, increases the activities of enzymes involved in β-oxidation. Prabhakar et al. found that α-amyrin significantly increased the mRNA expression and protein level of PPAR-α, leading to increased lipid metabolism and deposition in the liver.¹²⁹ Ardiansyah et al. also indicated that lupeol improved lipid metabolism by decreasing the mRNA expression of the genes involved in lipid synthesis in the liver.¹³⁰ But the function was independent of the benefits in blood pressure amelioration. Both lupeol and its ester ameliorated the hypercholesterolemic condition, while lupeol linoleate was more effective.¹⁴ The mechanism might be due to the reduced absorption and enhanced excretion of cholesterol.¹³¹

Daisuke et al. indicated that the 24-methylene cycloartanol and cycloartenol prevented obesity through controlling the release of glucose-dependent insulinotropic polypeptide (GIP).¹³² The supplementation with cycloartenol and 24-methylene cycloartanol caused less body weight gain, promoted fat utilization, and enhanced the expression of genes associated with lipid oxidation, whereas it suppressed the gene expression related to lipid synthetase and sterol regulatory element-binding protein (SREBP)-1c. A later study also revealed that the 24-methylene cycloartanol and cycloartenol decreased the postprandial hyperglycemia by decreasing glucose absorption in the intestine both in mice and humans.¹³³ In addition to the abovementioned 4,4-dimethyl phytosterols, lanosterol at a 1% dose did not show any significant effect on serum lipid profiles, while the HDL-C level was significantly upregulated at a 2% dose.¹⁰⁷ Moreover, taraxerol restored the abnormal changes in type 2 diabetes.⁹⁹ Overall, the proposed underlying mechanism of the anti-hyperglycemic and anti-hypolipidemic effects in obesity-related metabolic disorders is presented in Fig. 3.

Fig. 3 Proposed mechanism underlying the anti-hyperglycaemic and anti-hypolipidemic effects by 4,4-dimethyl phytosterols. Obesity is commonly caused by a combination of excessive food intake, insufficient physical activity, and genetic susceptibility. 4,4-Dimethyl sterols exert a potential efficacy against obesity through multiple mechanisms involved in heart protection, lipid metabolism, adipocyte response, and gut functions. Total cholesterol, TC; total triacylglycerol, TG; very low-density lipoprotein, VLDL; low-density lipoprotein cholesterol, LDL-C; high-density lipoprotein cholesterol, HDL-C; free fatty acid, FFA; cholesterol ester hydrolase, CEH; lecithin/cholesterol acyl transferase, LCAT; cholesterol ester synthase, CES; lipoprotein lipase, LPL; peroxisome proliferator activated receptor-α, PPAR-α; 3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGCR; fatty acid synthase, FASN; acetyl-coenzyme A carboxylase α, ACACA; sterol regulatory element binding protein-1c, SREBP-1c; sterol regulatory element binding protein-2, SREBP-2; steroid binding protein, SBP; interleukin-6, IL-6; tumor necrosis factor-α, TNF-α; and monocyte chemoattractant protein-1, MCP-1.

3.5. Antinociceptive activity

A previous study showed that euphol prevented the mechanical nociceptive activity induced by ligation through decreasing the level of cytokines.¹³⁴ The proposed mechanisms revealed by Dutra et al. include: (i) modulation of the protein kinase C epsilon (PKCε) pathway; (ii) activation of NF-κB and CREB; and (iii) production of algesic proteins and pain mediators.¹³⁵ In another study, lupeol was found to exhibit antinociceptive effects that can be used against inflammatory pain, but not post-operative pain, through the reduction of IL-1β and TNF-α.¹³⁶ Interestingly, toxicity studies of lupeol indicated that no systemic toxicity and no central depressant symptom or mortality were observed even at the highest effective dose (100 mg kg⁻¹) for 7 days.^137,138 However, compared to indomethacin, lupeol did not exhibit antinociceptive activity.¹³⁹ This result might suggest a different antinociceptive mechanism between lupeol and indomethacin.

Oliveira et al. utilized two models of visceral nociception to evaluate the responses after oral pretreatment with α,β-amyrin. The mixture neither altered the anesthesia time, nor affected the motor coordination.^140,141 Thus, the antinociceptive mechanism of α,β-amyrin is probably involved in the opioid system and the vanilloid receptor (TRPV1). Similarly, the 24-methylene cycloartanol produced a marked and dose-dependent reduction of acetic acid-induced abdominal constriction. The function was also through a coordination with the opioid system, but not an interaction with a peripheral or central depressant.¹⁴² Otuki et al. found that α,β-amyrin given by several routes prevented the capsaicin-induced neurogenic nociception, and indicated that α,β-amyrin exerted an effective antinociceptive activity, particularly the inflammatory nociception.⁹³ The mechanism seems to be related to the regulation of the protein kinase A- or protein kinase C-sensitive pathway. These results presented herein suggest that 4,4-dimethyl phytosterols exhibit potent antinociceptive activity, hence constituting a promising perspective to pharmacological development.

3.6. Other functions

Except for the abovementioned benefits, 4,4-dimethyl phytosterols have been reported to exhibit other pharmacological activities, such as anti-cataract,^143,144 anti-fertility,^145,146 anti-microbial,^147,148 anti-depressant,¹⁴⁹ and anti-viral activities.¹⁵⁰ Lupeol pretreatment could decrease the level of malonaldehyde (MDA) and reactive oxygen species (ROS) in lenses, which indicated that the anti-cataract benefit might be attributed to the antioxidant activities.¹⁴³ Additionally, lanosterol was proved to be effective for the prevention of protein aggregation, and may be developed as a promising therapeutic strategy for cataract.¹⁴⁴

In addition to cataract, Gupta et al. demonstrated that lupeol and amyrin in their acetate form caused a significant decrease in the weights of reproductive organs. Sperm motility and sperm density were also decreased, leading to a complete prevention of fertility.^145,146 To further investigate the anti-fertility activity, Mannowetz et al. found that lupeol regulated the activation of the calcium channel of sperm (CatSper) via the modulation of α/β hydrolase domain-containing protein 2 (ABHD2).¹⁵¹ ABHD2 is an important enzyme appearing in the endogenous cannabinoid system (ECS). Recent literature reports tentively discovered that a potential linkage existed between the benefits of 4,4-dimethyl phytosterols and the ECS, which is discussed in the following section.

4. Exploring the linkage between 4,4-dimethyl sterols and the ECS

4.1. A glimpse of the ECS

Numerous studies have been conducted to clarify the biological role of the ECS, regarding its function in NCDs, and the potential of its pharmacological exploitation. The ECS comprises (i) two G protein coupled cannabinoid receptors, CB1R and CB2R; (ii) their ligands, which are lipid molecules, 2-arachidonoylglycerol (2-AG) and N-arachidonylethanolamide (AEA); and (iii) the enzymes that regulate endocannabinoid biosynthesis and degradation, such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL).^152,153 AEA is produced via phospholipase D (PLD)-mediated hydrolysis of N-arachidonoyl phosphatidylethanolamine, and degraded by the FAAH,¹⁵⁴ while 2-AG is synthesized via diacylglycerol lipase (DAGL)-mediated hydrolysis of diacylglycerol (DAG), and degraded by MAGL.¹⁵⁵ Other serine hydrolases were also involved in the degradation of 2-AG, such as ABHD2.¹⁵⁶

CB1R is intensively expressed on both glutamatergic and GABAergic interneurons in the peripheral and the central nervous systems, and has been widely followed in the maintenance of homeostasis in health. But postsynaptic localization for CB1R was also indicated.^157,158 They are accountable for the psychoactive properties induced by tetrahydrocannabinol (Δ9-THC), and are critical in analgesic action and energy regulation. Furthermore, CB1R also exists in microglia, astrocytes and oligodendrocytes for the control of inflammatory reaction and further participates in the role in the nervous system.¹⁵⁹ Recently, the alteration of the ECS or over-expression of CB1R was associated with various diseases, such as inflammatory diseases, obesity, diabetes and Alzheimer's disease.¹⁶⁰ 4,4-Dimethyl phytosterols have been attentively illustrated to regulate the ECS, thus exhibiting many beneficial functions.

4.2. ECS ameliorated by 4,4-dimethyl phytosterols

Euphol was the first 4,4-dimethyl phytosterol discovered to exert beneficial effects on the ECS. King et al. screened various commercial chemical compounds and found that euphol inhibited MAGL through interacting with a novel regulatory region in a reversible behavior.¹⁶¹ A later study found that euphol exerted an anti-inflammatory function by ameliorating the ECS. Euphol inhibited mechanical hyperalgesia, and it did not cause any side effects in the central nervous system.¹³⁴ However, the antagonists of CB1R or CB2R, and the knockdown gene of CB1R and CB2R, significantly blocked the beneficial effect.¹³⁴ Similarly, pretreated α,β-amyrin together with the antagonists or knockdown gene of the cannabinoid receptors also significantly prevented their anti-nociceptive effect.¹⁶² Thus, the literature revealed that euphol and α,β-amyrin could directly activate CB1R and CB2R. To further investigate how α-amyrin and β-amyrin were coordinated with the ECS, a colitis mouse model was utilized by Matos et al. They showed that α,β-amyrin significantly inhibited colonic damage, and the pro-inflammatory mediators.¹⁶³ But different from the results obtained by Dutra et al.¹³⁴ and Silva et al.,¹⁶² Matos et al. found that the pharmacological blockade of CB1R, but not CB2R, prevented the beneficial activity of α-amyrin and β-amyrin. Besides, the expression of MAGL and FAAH was significantly decreased with the pretreatment of α,β-amyrin.¹⁶³ Based on these results, the study conducted by Chicca et al. examined the binding interactions between cannabinoid receptors and α,β-amyrin using several cells and purified enzymes.¹⁶⁴ They found that β-amyrin was more potent than α-amyrin, and exerted its analgesic functions via inhibiting the endocannabinoid compound without directly interacting with endocannabinoid receptors. Meanwhile, it was deduced that the geminal methyl groups of β-amyrin in the A ring were more effective than the vicinal methyl groups of α-amyrin. β-Amyrin inhibited 2-arachidonoylglycerol (2-AG) hydrolysis without directly targeting cannabinoid receptors.¹⁶⁴

Additionally, lupeol was discovered to inhibit ABHD2 activity. CatSper played a significant role in the motility and fertility of sperm.¹⁶⁵ Lupeol could activate CatSper of sperm through binding with ABHD2, and thus could be utilized as a contraceptive compound for fertilization prevention.¹⁵¹ As for the activity evaluation, a recent study proved that α-amyrin and β-amyrin potently decreased the MAGL activity in vitro, but the difference of IC₅₀ was not statistically significant.¹⁶⁶ Previous studies found that α-amyrin and β-amyrin were comparable to synthetic compounds,¹⁶² and α,β-amyrin was 200–300 fold more potent than Δ9-THC.¹⁶⁴ Based on these reports, the endowed health benefits of the ECS involved in 4,4-dimethyl phytosterols are summarized in Table 3 and Fig. 4.

Fig. 4 The ECS involved in 4,4-dimethyl phytosterols endowed healthy benefits in the body. The cannabinoid system involves the release, transport and degradation of various endocannabinoid compounds, such as 2-AG or 2-OG mediated by MAGL, and AEA or OEA mediated by FAAH. The 4,4-dimethyl sterols can interact with CB1R, thus regulating the endocannabinoids in different tissues. In the spinal cord, euphol inhibits MAGL activity and up-regulates 2-AG, which binds to CB1R further inhibiting the PKCε pathway, thus decreasing the activation of NF-κB, CREB, and COX-2 production. In the colon, α,β-amyrin inhibited the activity of MAGL and FAAH by modulating CB1R activation, thus reducing the pro-inflammatory cytokines and expression of the adhesion molecule mRNA. In the brain, β-amyrin exerts beneficial effects by inhibiting 2-AG hydrolysis, but not FAAH. Whether the 4,4-dimethyl sterols could inhibit FAAH activity remains to be confirmed. N-Arachidonylethanolamide, AEA; oleoylethanolamide, OEA; cannabinoid receptor 1, CB1R; fatty acid amide hydrolase, FAAH; monoacylglycerol lipase, MAGL; 2-arachidonoylglycerol, 2-AG; 2-oleatemonoglycerol, 2-OG; cyclooxygenase-2, COX-2; protein kinase C epsilon, PKCε; adenosine triphosphate, ATP; protein kinase A, PKA; cyclic-adenosine monophosphate, cAMP; adenylate cyclase, AC; nuclear factor-κB, NF-κB; cyclic AMP response element-binding protein, CREB; interleukin-6, IL-6; intercellular adhesion molecule 1, ICAM-1; myeloperoxidase, MPO; tumor necrosis factor-α, TNF-α; N-acetylglucosaminidase, NAG; interleukin-1β, IL-1β; interleukin-4, IL-4; interleukin-6, IL-6; platelet cell adhesion molecule 1, PCAM-1; inducible nitric oxide synthase, iNOS; keratinocyte-derived chemokine, CXCL1/KC; arachidonic acid, ARA; prostaglandin E2, PGE2. (+), promotion; (−), downregulation; (⊢), inhibition; (↑), increase; (↓), decrease.

Table 3 Health effects of 4,4-dimethyl phytosterols and the ECS involved

Compound	ECS response	Model	Description	Ref.
Monoacylglycerol lipase, MAGL; cannabinoid receptors, CB; cannabinoid receptor 1, CB1R; cannabinoid receptor 2, CB2R; 2-arachidonoylglycerol, 2-AG; fatty acid amide hydrolase, FAAH; dextran sodium sulfate, DSS; and α/β hydrolase domain-containing protein 2, ABHD2.
Euphol, 30 and 100 mg kg^−1 p.o.	Interaction with CB1R and CB2R	CB1R and CB2R knockdown mice	Anti-nociceptive effect was reversed by pre-treatment with either CB1R or CB2R antagonists, as well as the knockdown gene of CB1R and CB2R.	134
Lupeol, IC50 109 nM	ABHD2 activity↓	Purified human spermatozoa	Significantly reduced the activation of the calcium channel of sperm by either progesterone or pregnenolone sulfate.	151
Euphol, 10 μM	MAGL activity↓	Rat neuron culture	Inhibited MAGL activity with high potency through a reversible mechanism.	161
α,β-Amyrin, 30 mg kg^−1 p.o.	Activation of CB1R and CB2R	Mice and rats	Directly bound with high affinity to CB1R and lower affinity to CB2R.	162
α,β-Amyrin, 10 mg kg^−1, i.p.	MAGL activity↓ FAAH activity↓	DSS-induced colitis in mice	mRNA expression for MAGL and FAAH was significantly reduced in the colon. CB1R, but not CB2R, pharmacological blockade reversed the beneficial effects.	163
β-Amyrin, 1 and 10 μM	2-AG content↑ MAGL activity↓	CHO-K1 cells, BV2 cells, pig brain	Inhibited the degradation of the endocannabinoid 2-AG without interacting directly with CB receptors.	164
α,β-Amyrin, lupeol	MAGL activity↓	MAGL assay in vitro	Relative potency of MAGL inhibition as determined by IC50 was as follows: α-amyrin (487.4 ng mL^−1), β-amyrin (676.7 ng mL^−1) and lupeol (785.4 ng mL^−1).	166

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Overall, 4,4-dimethyl phytosterols offer broad and largely unexplored benefits for preventing various diseases and provide new insights into the underlying relationship with the ECS. The identification of the core region of MAGL provided a target for the characterization of potent inhibitors. King et al. revealed that a common pocket in euphol was found to be in the α-helical lid domain of MAGL, and it interacts reversibly with neighboring Cys201 and Cys208 present at the border of such a pocket.¹⁶¹ Although the observed activity through the interaction via MAGL offered an explanation of the ameliorating activity, the detailed mechanism needs further investigation. Recently, it has been demonstrated that the sterol A-ring plasticity is a critical factor for the governing hydrophobic interaction and molecular recognition substrate.

Sterol A-ring variants exhibit various substrate activities. Among all the assayed variant structures, contracted A-ring sterol exerts the highest activity.¹⁶⁷ The 4,4-dimethyl phytosterols possess two methyl substituents at the carbon-4 position of the A-ring. The methyl substituent is attached with great significance in the rational drug design. The increased lipophilicity by methylation can drastically change the bioavailability and thus the efficacy of a bioactive molecule and its mode of interaction with the bioreceptors.¹⁶⁸ A previous study suggested that the introduction of a methyl group to imidazolinediones increased the lipophilicity and ability to dislocate rimonabant (SR-141716A), which was a specific antagonist for CB1R.¹⁶⁹ In addition, 4,4-dimethyl substituents could also influence the physical properties of biological membranes. The molecular structure of a sterol determined the ability to induce phase separation and the curvature of the formed liquid-ordered phase. In particular, the methylated sterols were able to induce domain formation, which might play an important role in the cell-biological processes.¹⁷⁰ For example, increasing the methyl groups at the carbon-4 position would render the phytosterol less efficient in lowering glucose permeability.¹⁷¹ Another important effect of 4,4-dimethyl groups may rely on severe steric hindrance to the 3β-OH group of sterols. In the biological membrane, a 3β-OH group seems to be a prerequisite for a specific interaction in the membrane.¹⁷²In vitro chemical assays indicated that the steric hindrance caused by the presence of ortho-methyl groups could influence the hydrogen-bonding interactions.^173,174 The steric hindrance caused by the extra methyl group stabilized the hydrogen bonding network, and prevented the ionization of the neighboring amino acid residues. A extra methyl group forced OH to rotate and donate a hydrogen bond to other functional residues.¹⁷⁵ Thus, the methyl groups in the A-ring might play an important role in the function of 4,4-dimethyl phytosterols, while further confirmation is still necessary.

5. Clinical trials

To test the clinical utility of 4,4-dimethyl phytosterols in treating diseases in humans, the unsaponifiable fraction from unripe fruits of Olea europaea, containing the maximal content of 4,4-dimethyl phytosterols (lanosterol, cycoartanol, and α,β-amyrin), was utilized in a small pilot clinical study with five patients affected by osteoarthrosis. According to the clinical results, treatment with the ointment prevented the hand and knee inflammatory symptoms in all patients without any adverse reactions.¹⁷⁶ In another trial, Okahara et al. proved that the consumption of brown rice with a high content of cycloartenol and 24-methylene cycloartanol led to a lower postprandial hyperglycemia.¹³³ The postprandial blood glucose level was significantly decreased by treatment with 24-methylene cycloartanol- and cycloartenol-enriched rice. Additionally, lupeol has been widely studied in clinical trials. Twenty-eight patients were enrolled in a non-randomized pilot study to evaluate lupeol in treating actinic keratosis, which may develop into skin cancer if left untreated. More than 75% of the injuries were cured by a lupeol-containing birch bark ointment.¹⁷⁷ Currently, clinical trials conducted by Seoul National University Hospital,¹⁷⁸ have already indicated that 2% of lupeol cream could treat patients suffering from mild–moderate face acne. In another case, lupeol promoted the focal lysis of tumors when injected at primary sites. Administration of lupeol treated malignant melanomas successfully.¹⁷⁹

However, excess accumulation of 4,4-dimethyl phytosterols might cause various malformation syndromes. For example, lanosterol is the precursor substance of cholesterol, which is the first sterol in the cholesterol synthesis pathway. Malformation syndromes caused by inborn errors of cholesterol synthesis have been reviewed in detail.^180,181 Additionally, Fang et al. proved that 2 μg ml⁻¹ lanosterol not only failed to support the growth of auxotrophic mammalian cells, but also killed wild type mammalian cells.¹⁸² A recent study also suggested a moderate toxic potential for lupeol by targeting to the estrogen-receptor (ERα) involved in fertility, which could trigger undesired adverse effects.¹⁸³ Therefore, the safe dose and long-term side effects warrant further investigation before these 4,4-dimethyl phytosterols can be recommended as a treatment option.

Recent work showed that the ATP-binding cassette transporter (ABCA1) can efficiently efflux lanosterol. Lanosterol could be chosen as a preferred substrate for ABCA1. However, lanosterol was hardly a substrate of the acyl-coenzyme A:cholesterol acyltransferase (ACAT) because lanosterol contains the gem dimethyl groups, which could cause a severe steric hindrance to 3β-OH.¹⁸⁴ This property incapacitates the ability of ACAT1 to transport lanosterol in the endoplasmic reticulum. Considering that we can only take in phytosterols orally, there has been almost no literature about the absorption mechanism of other 4,4-dimethyl phytosterols. Therefore, further investigation is necessary to study the related mechanisms.

6. Future directions

The clinical trials demonstrated that the 4,4-dimethyl phytosterols can be exploited as prospective targets for the prevention of various diseases. However, many considerations still require further exploration, such as the appropriate dose, duration of exposure, and relative bioavailability. Most of the studies have attempted to show the benefits by using animal models. It seems that there still exists a research gap in investigating in humans when applied as functional foods or nutraceuticals. Therefore, in addition to animal models, future studies should be directed towards the clinical evaluation of 4,4-dimethyl phytosterols. A collaboration between food scientists and medical researchers is recommended. Furthermore, research is still needed to confirm the potential relationship between 4,4-dimethyl phytosterols and the ECS, as well as the detailed mechanisms. For example, the 4,4-dimethyl phytosterols are effective against PD, and future studies should focus on whether the anti-Parkinsonian effect is a direct consequence of ECS-mediated mechanisms, or simply the result of other functions, such as anti-inflammatory activity.

7. Summary

4,4-Dimethyl phytosterols are a class of bioactive compounds commonly found in plants and vegetable oils, which are suitable sources for convenient extraction or dietary consumption. From the above discussion, it is obvious that the naturally occurring 4,4-dimethyl phytosterols show great promise in the fight against various diseases such as inflammation, Parkinsonism and Alzheimer's disease, cancer, cataract, etc. The in-depth mechanism is likely to involve the ECS. Moreover, the clinical studies of 4,4-dimethyl phytosterols were further reviewed to support their potential development as therapeutic agents for multiple diseases. However, there still exists a research gap in exploring the benefits of 4,4-dimethyl phytosterols in humans when applied as functional foods or nutraceuticals. Thus, more clinical studies should be considered in the future.

Author contributions

Qingzhe Jin and Hui Zhang contributed to the conception of the manuscript. Tao Zhang and Ruijie Liu drafted the manuscript. Ming Chang and Xingguo Wang critically revised the manuscript and gave the final approval.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The National Natural Science Foundation of China (Grant No. 31972037 and 31872895), and the National First-Class Discipline Program of Food Science and Technology (JUFSTR20180202) are acknowledged. We thank Prof. J. Thomas Brenna for his linguistic assistance during the preparation of this manuscript. Dr Tao Zhang particularly desires to thank Ms Yingying Wang for her meticulous and continuous support.

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