Studies of the Immune Enhancement Properties of Black Raspberry Seed Oil

Background
Free radicals are atoms or molecules that are highly reactive with other cellular structures in the human body due to an unpaired electron. Consequently, free radicals are capable of chemically altering nearly all major classes of biomolecules (e.g., lipids, nucleic acids, proteins). Free radicals can be produced in vivo by factors such as dietary imbalances, tobacco smoke, pollutants, or from sources such as inflammation and biochemical reactions. ^1-3 They are capable of damaging DNA, inhibiting its repair, and increasing the probability of contracting a viral infections or cancer. ^4-5

Interestingly, free radicals are also generated by cells of the immune system and their purpose is to destroy invading organisms. However, the presence of these strong oxidants places additional stress on the immune system (called “oxidative stress”), which can result in a diminished response against invaders. Furthermore, the long-term presence of these oxidizing species will eventually be detrimental to the human body.⁶

Oxidative stress is thought to play an important role in the pathogenesis of numerous chronic diseases, such as coronary heart disease and cancer. Although there are many factors in the development of these diseases, considerable experimental evidence has linked the production of free radicals to biological damage that can provide a basis for the initiation and progression of certain diseases.^7-10

Humans and other aerobic organisms have evolved a variety of mechanisms to protect themselves from the deleterious effects of free radicals. The body’s defense systems, using certain enzymes, protect against free radical damage either directly or indirectly. Water and lipid-soluble antioxidants (such as ascorbate (vitamin C), alpha tocopherol (vitamin E), and beta carotene) also act to eliminate free radicals.^7-8 These antioxidants help shield DNA from the deleterious effects of oxidative damage by absorbing unstable oxygen molecules.¹²

A number of sources indicate a relationship between diet and cancer incidence in humans.¹³ The geographic distribution of certain types of cancer, the changing cancer patterns, and data from experimental animal studies all indicate that diet and nutrition are important factors in the control and prevention of human cancers.¹⁴ The potential importance of diet in cancer prevention is also noted by the suspected causes of some cancers. Most cancers (an estimated 80% to 90%) have environmental causes and are therefore potentially preventable. Much controversy surrounds the actual percent of cancers associated with dietary factors, but it has been estimated that in men 30% to 40% of all cancers are in some way related to diet. In women, it is believed that 60% of all cancers are related to diet. Another study estimates that 35% of cancer is diet related.^15-17 Regardless of the exact numbers, these are impressive percentages.

Antioxidants
Presumably, pathology due to oxidative stress results when the generation of free radicals exceeds the cell’s capacity to protect or repair itself. Therefore, if oxidative damage is an important etiologic factor in the pathogenesis of diseases such as cancer, then it follows that antioxidants, which act to reduce oxidative stress, may play a role in the prevention or treatment of these diseases. The accumulation and growth of free radicals in tissues is often found in association with suppressed immune function, including infections such as HPV and HIV, cancer, and heart disease.^18-21 In fact, damage to heart blood vessels and the incidence of coronary heart disease has been shown to be reduced with increased dietary antioxidant intake.²² The protective effects of topical antioxidants (vitamin A derivatives such as retinoic acids) against proliferative dermatological diseases as well as photo-aging have been well documented.^23-25 Many studies continue to demonstrate below normal antioxidant tissue and blood plasma levels in women with HPV and other cervical neoplasms, while high levels provide protection against their initiation and progression.^26-27

Like the B vitamins, the beneficial effects of antioxidants are most notable when combined with one another. In fact, diets high in antioxidants (e.g., the traditional Greek Mediterranean diet) have been shown to be protective against cancer and various diseases.²⁸ It is believed that antioxidants alter cancer incidence and growth by acting as anti-carcinogens. Nutritional anti-carcinogens function by (a) inhibition of tumor initiation via alteration of cellular metabolism, (b) picking up active forms of carcinogens and preventing them from reaching target sites, (c) alteration of the body’s defense systems, (d) inhibition of cancer progression once it has been initiated by the alteration of cell differentiation, and (e) prevention of gene activation and cellular proliferation by tumor promoters.^14-16

Plant Seed Oils
Plant seed oils that can be extracted from the fruit, leaves, or seeds of various plants have been found to be an excellent source of antioxidants. In addition to traditional antioxidants such as vitamins C and E, plant oils contain phenolic compounds which are excellent free radical scavengers due to their electron structure. Recent studies have shown that plant oils rich in antioxidants also increase cellular immune response.^6-32

Laboratory Study
For over a decade, we have been investigating methods to treat liver cancer (adenocarcinoma that metastasized from the colon) in mice in our laboratory. We postulated that enhancing the mouse’s immune system with antioxidants may improve clinical success of the cancer treatment methodology we were researching. Certain plant seed oils are extraordinarily high in antioxidants and, for the purpose of our study, black raspberry seed oil was used as a dietary supplement to provide the mice with a source of a variety of antioxidants because it has one of the highest levels of antioxidants. Moreover, black raspberry seed oil has a higher oxygen radical-reducing capacity than palm, corn, sunflower or olive oil.³⁴

The oil was obtained from the black raspberry seeds via the cold press manufacturing method. The cold press extraction method yields excellent quality oil with minimal chemical decomposition compared to traditional high temperature extraction methods.³⁵ When using cold-pressing, nothing is added to the seeds or to the oil to increase production during the manufacturing process. The oils are pure and unadulterated.

In the study, black raspberry seed oil was added daily to the food of the mice and the amount of oil administered was equal to 10% of the rodent’s weight. After varying periods of time, the mice were euthanized and their body chemistry carefully examined.

In the course of this research, it was discovered that the highly potent antioxidant oil, black raspberry seed oil, appeared to augment the populations of both NK (natural killer cells) and CD8(+) T (cytotoxic T lymphocyte) cells. This research is key because an increase in the number of NK and CD8(+) cells enables to body to better fight the damage caused by free radicals and enhance the body’s ability to fight disease and cancer.

THE STUDY:

Evaluation of splenic lymphocyte populations when black raspberry seed oil is administered

EXPERIEMENTAL DESIGN:

4 groups of mice were used at 6-8 weeks of age

Day 0
Group 1 - standard rodent meal
Group 2 - standard rodent meal + 10% black raspberry oil

Day 3, 7, or 14
Mice will be sacrificed for spleen harvest and to obtain cardiac blood samples. Spleens will be processed for flowcore analysis and the data will be processed using StatView. Cardiac blood will be centrifuged to collect serum for ELISA analysis of circulating cytokines at a later date.

RESULTS/SUMMARY:
Recent studies have shown that plant oils rich in antioxidants also increase cellular immune response. We postulated that the addition of antioxidant oil to a murine diet would further elevate increases NK cells and CD8+ lymphocytes. To test this hypothesis, animals were randomly placed into two groups: diet with and without oil. For the duration of each study, the animals were fed standard rodent chow or standard rodent chow supplemented with 10% (by weight) black raspberry seed oil, a potent antioxidant plant oil. Splenic lymphocyte populations were analyzed 3, 7, and 14 days. We found significantly elevated CD8+ lymphocyte populations in the oil group at all time points when compared to the other group. Furthermore, we observed that the NK population peaked on day 7. These results suggest that antioxidant oil in conjunction with the normal diet augments cytotoxic immune response.

TABLE 1.

EXPERIMENTAL DESIGN
	Without Oil	With Oil
Diet	Standard rodent diet	Standard rodent diet + 10% black raspberry seed oil by weight

FIGURE 1. Effect of black raspberry seed oil diet on natural killer (NK) cell populations. After splenocytes were isolated, flow cytometry was utilized to determine the percentage of NK cells present. Each value represents the mean ± SD of at least 5 mice. Error bars are not apparent if less than 0.5.

TABLE 2.

FIGURE 2. Effect of black raspberry seed oil diet on cytotoxic T lymphocyte (CD8+) populations. After splenocytes were isolated, flow cytometry was utilized to determine the percentage of CD8+ lymphocytes present. Each value represents the mean ± SD of at least 5 mice. Error bars are not apparent if less than 0.5.

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